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From the * Department of Genetic Medicine, Weill
Medical College of Cornell University, New York, New York;
Department of Biochemistry, Molecular Biology
and Cell Biology, Northwestern University, Evanston, Illinois;
Department of Neurobiology and Physiology,
Northwestern University, Evanston, Illinois; and
Department of Cell Biology, University of
Virginia, Charlottesville, Virginia.
| Correspondence to: Dr Erwin Goldberg, Department of Biochemistry, Molecular Biology and Cell Biology, 2205 Tech Dr, Northwestern University, Evanston, IL 60208 (e-mail: erv{at}northwestern.edu). |
| Received for publication October 21, 2005; accepted for publication January 16, 2006. |
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Abstract |
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Key words: Ovary, gene expression, glycolysis, enzyme
Three genes, Ldha, Ldhb, and Ldhc, encode the A, B, and C subunits (1, 2, and 3, respectively, in terminology applied to murine cells) that comprise the LDH family of enzymes. Generally, the isozyme composition of a particular tissue is characteristic of the metabolic requirements of that tissue. For example, so-called aerobic tissues show an abundance of Ldh2, whereas more anaerobic requirements are met by Ldh1 (reviewed in Everse and Kaplan, 1975). Ldh3 predominates in male germ cells, presumably to support energy production in spermatids that favor lactate as substrate and in spermatozoa with a characteristic aerobic glycolytic path to yield adenosine triphosphate (ATP) (Goldberg, 1972). The importance of glycolysis to motility was rediscovered recently by Mukai and Okuno (2004), who showed that inhibition of oxidative phosphorylation had little effect on sperm ATP levels or flagellar activity. Miki et al (2004) disrupted by gene targeting glyceraldehyde 3-phosphate dehydrogenase-S, a mouse gene expressed only during spermatogenesis and a key glycolytic enzyme. In their study, males were infertile, sperm motility was sluggish without forward progression, and ATP levels were markedly reduced, though mitochondrial oxygen consumption was unchanged. Duan and Goldberg (2003) reported that inhibition of Ldh3 with the substrate analogue oxamate blocked sperm motility and capacitation. Thus, the properties of Ldh3 are well suited to testis and sperm function. Ldh2 is the most abundant LDH isozyme on the oocyte (Brinster, 1968; Roller et al, 1989). However, we recently identified Ldh3-specific peptides during analysis of the mouse oocyte proteome, suggesting that this isozyme is expressed in both male (Wheat and Goldberg, 1983; Millan et al, 1987) and female germ cells. This report documents these findings and further investigates Ldh3 expression and subcellular localization of the isozyme in the oocyte and early embryo. Surprisingly, we find that Ldh3, likely of maternal origin, is detected in early embryos until the blastocyst stage of development, raising the possibility that this molecule may function as the product of a maternal effect gene.
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Materials and Methods |
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Collection and Indirect Immunofluorescence Analysis of Eggs and Embryos
Protocols for the use of animals in these experiments were approved by
Cornell Medical College, the University of Virginia, and Northwestern
University Animal Care and Use Committees and were in accordance with the
National Institutes of Health's standards established by the Guidelines for
the Care and Use of Experimental Animals. All oocytes and embryos were
obtained from ICR 25- to 30-g females. Germinal vesicle (GV) oocytes were
collected by follicular puncture as described previously (Coonrod et al,
1999,
2001). Metaphase II (MII) eggs
were isolated from the oviducts of superovulated female mice. Pronuclear
zygotes, 2-cell, 4- to 8-cell, morula, and blastocyst embryos were isolated
from the oviducts and uterus of superovulated and mated mice at the
appropriate times. Upon collection, oocytes and embryos were fixed immediately
in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 minutes at
room temperature. After fixation, oocytes and embryos were washed 5 times in
immunofluorescence (IF) media (PBS + 1% bovine serum albumin [BSA] plus 0.5
normal goat serum [NGS]) and then permeabilized with 0.5% Triton-X 100 in PBS
for 30 minutes. Oocytes and embryos were then washed 5 times and incubated
with anti-Ldh3 or with an absorbed anti-Ldh3 immune antisera adjusted to the
same protein concentration (3.6 µg/mL) in IF media overnight at 4°C.
Oocytes and embryos were washed 5 times and incubated for 3 hours at room
temperature with donkey anti-rabbit fluorescein isothiocyanate–labeled
secondary antibody (Jackson Immunoresearch) and Hoechst to stain the DNA.
Finally, oocytes and embryos were washed, mounted on slides, and visualized at
1000 x under a Zeiss Axiovert-200 fluorescence microscope and imaged
(Carl Zeiss, Inc, Thornwood, NJ).
RNA Isolation and Reverse Transcriptase Polymerase Chain Reaction
Mature female CD-1 mice were purchased from Harlan (Indianapolis, Ind) and
housed under constant environmental conditions with free access to mouse chow
and water. Mice were euthanized by CO2 inhalation followed by
cervical dislocation to ensure death. The ovaries were removed and cleared of
adherent connective tissue. GV oocytes and MII-arrested eggs were collected as
described above. Cumulus cells were collected and washed with PBS by
centrifugation at 10 600 x g. RNA was isolated from 50 oocytes
or eggs or from about 1 x 105 cumulus cells and extracted
with TRIZol reagent from Invitrogen (Grand Island, NY) according to the
protocol supplied by the manufacturer, with 10 µg glycogen used as carrier
to precipitate RNA. The RNA samples were incubated with Moloney murine
leukemia virus reverse transcriptase (Promega, Madison, Wis) and
oligo(dT)15 primer in a reaction volume of 25 µL, with 5 µL
used as the template for polymerase chain reaction (PCR). The DNA was
denatured at 94°C for 5 minutes, followed by 35 cycles at 94°C for 30
seconds, 60°C for 30 seconds, and 72°C for 60 seconds. An extension
time of 5 minutes at 72°C was added at the completion of the cycles.
Primers used for Ldh3 to yield a 357-bp sequence:
Primers used for Ldh3 to yield a 1048-bp sequence:
Primers specific to Ldh2:
Western Blotting
Oocytes or eggs were solubilized in the loading buffer for SDS-PAGE.
Ovarian tissue was homogenized in extraction buffer (1% Triton X-100; 150 mM
sodium chloride; 10 mM TrisCl (pH 7.4); 1 mM ethylene glycol bis-2-aminoethyl
ether-N,N',N'',n'-tetraacetic acid; 1 mM EDTA; 0.2 mM
Na3VO4; 0.2 mM phenylmethanesulfonyl fluoride; 0.5%
NP-40; 50 mM NaF; 5 mM benzimidine) and centrifuged for 10 minutes at 4°C
and 20 800 x g. The supernatant was recovered, and protein
concentration was measured with the Bio-Rad (Hercules, Calif) protein assay
kit. Proteins were separated on a 12% SDS-PAGE gel, transferred to
nitrocellulose membrane, blocked by 5% milk, and incubated with anti-mouse
Ldh3 antibody at 4°C overnight. The blots were incubated with secondary
antibody at room temperature for 1 hour and washed by TBST. Antibody binding
was visualized with the ECL kit from Pierce Biotechnology (Rockford, Ill).
Immunohistochemistry
Mouse ovaries were placed in 4% paraformaldehyde (Sigma, St Louis, Mo)
fixative at 4°C overnight. The tissue was dehydrated and paraffin
embedded. Four-micrometer microtome sections were obtained and mounted on
Superfrost-Plus slides (Vector Laboratories Inc, Burlington, Calif). For
immunohistochemistry, the slides were deparaffinized in xylenes and then
rehydrated for 3 minutes each in 100% ethyl alcohol (ETOH), 95% ETOH, 70%
ETOH, 50% ETOH, and ddH2O. Antigen retrieval was accomplished by
incubating slides in 10 mM sodium citrate and heating in a microwave oven on
high for 2 minutes and on low for 8 minutes. The slides were cooled in sodium
citrate solution for 20 minutes, washed in TBS-T (Tween) to permeabilize, and
then incubated in 3% hydrogen peroxide in TBS for 15 minutes. Endogenous
avidin and biotin was blocked using the Avidin-Biotin Blocking Kit (Vector)
for 15 minutes each. The sections were blocked for 1 hour in 10% serum (from
host of secondary antibody) in 3% BSA-TBS at room temperature and incubated
overnight in primary antibody (anti-Ldh3) diluted 1:1000 in the blocking
solution. Control slides were incubated in 1:1000 preabsorbed primary antibody
adjusted to the same protein concentration. The slides were rinsed in TBS-T
and incubated in secondary antibody (1:200 dilution) conjugated to biotin
(Vector) in 3% BSA-TBS for 30 minutes and rinsed in TBS-T before adding
avidin-biotin complex reagent (Vector) for 30 minutes. After rinsing in TBS-T,
diaminobenzidine substrate (Vector) was added for 3 minutes, and the reaction
was stopped by 5-minute incubation in ddH2O. The sections were
counterstained with hematoxylin. Immunohistochemical images were acquired on a
Nikon E600 microscope with a Spot Insight Mosaic 11.2 color digital camera
(Nikon, Huntley, Ill) and ADVANCED SPOT IMAGING software (Version 4.6,
Universal Imaging, Downingtown, Pa).
Enzymatic Activity Assay
Extracts of oocytes and embryos were prepared by gentle homogenization in
PBS, and equal amounts of protein were added to wells for separation by native
PAGE. The gel was incubated in reaction mixture for LDH activity as described
previously (Goldberg, 1963). In
this protocol, the gel is immersed in a reaction mixture containing lactate as
substrate; phenazine methosulfate to transport electrons; and nitro blue
tetrazolium, which precipitates when reduced to detect the LDH isozyme
positions on the gel. Ldh3 was purified from mouse testes by affinity
chromatography as described previously
(Goldberg, 1975).
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Results |
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32
kDa, indicated by an arrow in Figure
1) contained 3 peptides that identified the testis-specific
isozyme of lactate dehydrogenase (Ldh3: NCBI GenBank accession number
NM_013580) with the basic local alignment search tool. The peptides were
identical to the amino acid sequences 6-16, 42–57, and 232–245
(Figure 2). To confirm this
result, we used PCR to amplify the Ldh3 mRNA and immunofluorescence,
immunohistochemistry, and Western blotting with specific antibody to visualize
Ldh3. RNA isolated from GV oocytes and MII-arrested eggs was reverse
transcribed and analyzed by PCR. The 2 sets of primers were designed to
amplify a full-length transcript as well as a 357-bp fragment of the
transcript that would ensure specificity of the reaction. The results in
Figure 3A show a weak signal
amplified from Ldh3 mRNA that is present in GV oocytes but not in
MII-arrested eggs. The positive signal from testis extract was not detected in
heart muscle as the negative control.
Figure 3B shows the detection
by RT-PCR of Ldh2 mRNA in extracts of GV oocytes, MII-arrested eggs,
and 2-cell embryos. Figure 3C
presents the amplification with 2 sets of primers to confirm specificity of
amplification of Ldh3 mRNA from the GV oocytes. Cumulus cells were
negative. The sequence of the 357-bp and 1048-bp transcripts
(Figure 3C) was identical to
that in the GenBank for Ldh3. Immunolocalization of Ldh3 in oocytes
and preimplantation embryos is shown in
Figure 4. A specific signal was
obtained for all oocytes and preimplantation embryos evaluated. No signal was
observed in eggs and embryos that were stained with an aliquot of the
anti-Ldh3 immune sera that had been preabsorbed with Ldh3 peptide (a
representative preabsorbed oocyte is shown in
Figure 4; the staining pattern
in control embryos looked similar [data not shown]). Ldh3 mainly appears to
localize to the cortex of GV oocytes, MII-arrested eggs (MII oocyte), and
pronuclear-stage zygotes (PN zygote). However, staining for Ldh3 is also
present to a lesser extent throughout the cytoplasm in these cells. In 2-cell
embryos (2 cell), staining for Ldh3 is mostly limited to the cortical region
of each blastomere. Interestingly, however, no staining is seen in the
juxtaposed cortical regions of each blastomere. Ldh3 staining is also seen in
the cortex of the polar body, with the exception of the regions apposing the 2
blastomeres where no staining is seen. In 4- to 8-cell (4–8 cell)
embryos and morulae, staining for Ldh3 is seen in the peripheral cortical
regions of external blastomeres, whereas little staining is seen in cortical
regions where blastomeres are in apposition. The Ldh3 localization in oocytes
was performed with our Zeiss Axiovert-200 microscope, which is equipped with
Z-stacking capability. The images were obtained from a single section made
through the middle of the egg; therefore, it seems unlikely that the increased
fluorescence is due to an edge effect. Cortical and punctate cytoplasmic
staining can still be observed for Ldh3 in the blastocyst; however, staining
levels appear to be reduced by this stage. The light image shows the focal
plane in which the image was captured. Although there appears to be a fairly
strong signal in these preparations, its amplification by the secondary
antibody provides only a qualitative and not quantitative picture of protein
in these cells. Histochemical analyses of ovarian sections
(Figure 5) show that primordial
and primary follicles do not make Ldh3 protein (panels A and B). As soon as
the follicle transitions from a primary to a secondary follicle, the oocyte
starts producing Ldh3. Panel D contains both negative primary follicles and a
positive early secondary follicle. As the follicle continues to grow, Ldh3
persists in the oocyte to the large antral follicles. For confirmation of Ldh3
in these tissues, extracts of testis, GV oocytes, MII oocytes, and heart
(negative control) were resolved by SDS-PAGE, blotted to nitrocellulose for
Western blots, and probed with specific antibody
(Figure 6). A positive signal
was obtained for testis, GV oocytes, and MII oocytes but not for heart.
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We were unable to detect Ldh3 enzymatic activity in egg extracts resolved by native PAGE followed by incubation in substrate-coenzyme reaction mix, though Ldh2 activity was high (data not shown). Although the full-length protein is present, detection of activity is likely obscured because of the relatively low level of Ldh3 in these extracts and the low sensitivity of the assay.
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Discussion |
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The original description of human LDH-C4 (Blanco and Zinkham, 1963; Goldberg, 1963) as a testis- or sperm-specific isozyme was based on enzymatic activity of protein bands separated by polyacrylamide and starch gel electrophoresis under nondenaturing conditions. The tissue specificity of LDH-C4 was established with electrophoretic techniques, antibody specificity, and immunofluorescence with tissue sections (reviewed in Goldberg and Wheat, 1976). Subsequently, by molecular cloning technology, Northern blotting confirmed the tissue specificity of Ldh3 gene expression (Millan et al, 1987). Additional confirmation was provided by experiments in which transgenes consisting of the Ldh3 promoter and LacZ were constructed (Li et al, 1998; Kroft et al, 2003). The ß–galactosidase reporter activity of several transgenes confirmed promoter activity in murine testes. It is interesting to note that we had observed Ldh3 in fertilized ova in earlier studies but attributed the immunohistochemical signal to LDH from supernumerary sperm (Bene and Goldberg, 1974) in the perivitelline space of the fertilized egg. In that study we did not detect antibody binding to unfertilized eggs. Presumably, antibodies would not penetrate live oocytes or embryos, and the present results were obtained with fixed tissues.
The current RT-PCR and immunohistochemistry data indicate that transcription and translation of Ldh3 occurs in the GV oocyte. The coincident localization of Ldh3 and ZP3 in oocytes in secondary-stage follicles (K. Tung, University of Virginia, personal communication) suggests that the Ldh3 gene is active during oogenesis. We were unable to detect Ldh3 mRNA beyond the GV arrested oocyte stage, suggesting that the antibody signal detected in Western blots and by indirect immunofluoresence is recognizing stable rather than newly synthesized Ldh3. The continued persistence of maternally derived Ldh3 in the early embryo is not surprising given that the stability of murine Ldh3 is well known and was, in fact, exploited during the purification of the protein (Goldberg, 1972, 1975). However, the protein detected could result from embryonic genome activation of Ldh3 expression. Embryos beyond the 2-cell stage were not assayed by RT-PCR. Interestingly, the striking cortical localization of Ldh3 in oocytes and early embryos is similar to that of 2 other abundant oocyte-restricted proteins, ePAD (Wright et al, 2003) and mPLA2g (Vitale et al, 2005). The similar expression and localization patterns raise the possibility that these maternal gene products may represent components of a larger complex yet to be defined.
Thus, Ldh3 appears first as a transcript and translation product in oocytes within secondary follicles, whereas the protein itself can be detected up to the preimplantation blastocyst. This raises perhaps the most pressing question, that is, whether or not Ldh3 has a function during oogenesis, oocyte maturation, or early development. Ldh2, composed of B subunits, has been described as a predominant LDH isozyme in eggs (Brinster, 1968; Roller et al, 1989) and would seem to be sufficient to satisfy the metabolic requirements of maturation and development. However, in its transit through the oviducts and uterus (where oxygen tensions may vary), the egg or embryo is likely to have periods when the anaerobic catalytic activity of Ldh3 is required. This prediction is supported by the observation that reproductive tract fluids are rich in glycolytic substrates including lactate (Gardner and Leese, 1990), and that from kinetic studies Ldh2 is sensitive to substrate inhibition (Everse and Kaplan, 1975), whereas Ldh3 is not inhibited by lactate (Goldberg, 1972). On the other hand, the abundance of Ldh2 for metabolism suggests that Ldh3 may play a different and perhaps noncatalytic role in oocytes that involves protein-protein or protein-nucleic acid interactions. The inability to detect Ldh3 enzymatic activity in the oocyte may simply be a sensitivity issue. The full-length coding sequence was detected (Figure 3C), so the catalytic tetramer should have been assembled. Finally, we cannot dismiss the possibility that Ldh3 transcription is a result of nonspecific gene activation. Whether this localization is biochemically relevant to egg metabolism or simply the result of random genetic instability remains to be established. It is perhaps instructive that ectopic expression of the human Ldh3 gene in a variety of tumor cell lines has been reported (Koslowski et al, 2002; Scanlan et al, 2004) and may result from aberrant demethylation of the gene or its transcriptional activators (Tang and Goldberg, unpublished observations).
In conclusion, previous findings supported the hypothesis that LDH-C4 (Ldh3) is a testis-specific isozyme and that Ldh3 expression is restricted to meiotic cells of the mammalian testis. Our finding that this gene is expressed in the oocyte was unexpected. Nevertheless, immunolocalization, RT-PCR, and Western blots provide solid evidence that the Ldh3 gene is transcribed and translated in oocytes and suggests that the previous paradigm on tissue distribution of Ldh3 now needs to be revised to include both male and female germ cells. It is important to report this result for inclusion in the large body of work on LDH-C4 as well as other presumed testis-specific genes.
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Footnotes |
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References |
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Goldberg E, Wheat TE. Induction of infertility in male rabbits by immunization with LDH-X. In: Spilman CH. Regulatory Mechanisms of Male Reproductive Physiology. Amsterdam, The Netherlands: Excerpta Medica; 1976: 133 –139.
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Vitale AM, Perlin JR, Leonelli L, Herr JC, Wright PW, Digilio LC, Coonrod SA. Mouse cPLA2gamma, a novel oocyte and early embryo-abundant phospholipase A2 gamma-like protein, is targeted to the nuclear envelope during germinal vesicle breakdown. Dev Biol. 2005; 282: 374 –384.[CrossRef][Medline]
Wheat TE, Goldberg E. Sperm-specific lactate dehydrogenase C4: antigenic structure and immunosuppression of fertility. In: Rattazzi MS, Scandalios JG, & Whitt GS eds. Isozymes: Current Topics in Biological and Medical Research. New York, NY: Alan R. Liss Inc; 1983: 113 –130.
Wright PW, Bolling LC, Calvert ME, Sarmento OC, Berkeley EV, Shea MC, Hao Z, Jayes FC, Bush LA, Shetty J, Shore AN, Reddi PP, Tung KS, Samy E, Allietta MM, Sherman NE, Herr JC, Coonrod SA. ePAD, an oocyte and early embryo-abundant peptidylarginine deiminase-like protein which localizes to egg cytoplasmic sheets. Dev Biol. 2003; 256: 74 –89.[CrossRef]
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