| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
The Jackson Laboratory, Bar Harbor, Maine 04609
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
developmental biology, follicular development, gamete biology, gametogenesis, oocyte development
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Another example of the metabolic support of oocytes by cumulus cells involves the uptake of some amino acids, such as L-alanine, which are poorly transported into mouse oocytes and require uptake first by cumulus cells and then transfer to the oocyte via gap junctions. When oocytes are cultured with radiolabeled L-alanine, the amount of radioactivity detected in the oocytes cultured enclosed by cumulus cells is greater than in oocytes cultured while denuded of cumulus cells [7]. Blocking the function of gap junctions in oocyte-cumulus cell complexes abrogates this difference [8]. We designate this group of amino acids as coupling dependent, coupling referring to gap junctional coupling between the oocyte and companion cumulus cells. Other (coupling independent) amino acids, such as L-leucine, are not differentially incorporated into cumulus cell-enclosed oocytes compared with denuded oocytes [7].
Suppression subtraction hybridization was used to identify transcripts expressed at higher levels in cumulus cells than in mural granulosa cells (MGCs); cumulus cell cDNA was used as the tester and mural granulosa cell cDNAs as the driver. The rationale for this project was that cumulus cell-enriched transcripts may be regulated by the oocyte. The transcript encoded by the solute carrier family 38, member 3 (Slc38a3) gene (MGI:1923507), was identified as enriched in the cumulus cell cDNA library. In the first part of this report, we describe the localization of Slc38a3 mRNA in the mouse ovary, and studies conducted to determine whether the elevated steady-state levels of this transcript in cumulus cells are maintained by oocytes. SLC38A3 is a sodium-coupled neutral amino acid transporter highly expressed in liver. Transporters in this family exhibit a preference for L-glutamate, L-histidine, and L-alanine. When Slc38a3 mRNA was injected into Xenopus oocytes, L-alanine transport into the frog oocytes was increased about 10-fold [9]. In the second part of this report, we determined whether oocytes affect the uptake of both coupling-dependent and independent amino acids by cumulus cells.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B6SJLF1 mice, bred and raised in the research colony of the investigators, were used for all experiments. Ovaries were obtained from 22-day-old mice either without gonadotropin priming or 44 h after priming by injection of 5 IU eCG to stimulate follicular development. The ovaries from 12-day-old mice were used for some experiments; no prior hormonal priming was used in this case. The mice were maintained according to the Guide for the Care and Use of Laboratory Animals (Institute for Learning and Animal Research).
In Situ Hybridization
After sequencing, a 332-bp clone from a cDNA library enriched in cumulus cell transcripts was identified as encoding a transcript of Slc38a3 encompassing exons 9 and 10. The clone was linearized with MaeIII (Roche Applied Science, Indianapolis, IN) to yield a 186-bp antisense RNA labeled with 33P CTP for in situ hybridization, which was carried out as we described previously [10] using ovaries from 12-day-old or 22-day-old mice 44 h after eCG priming.
Culture of Oocytes and Granulosa Cells
Cumulus cells and preantral granulosa cells were collected as previously reported [11]. Briefly, oocytes were surgically removed from granulosa cell-oocyte complexes either from antral follicles of 22-day-old primed mice or from preantral follicles of 12-day-old mice to collect either cumulus cells or preantral granulosa cells, respectively. These granulosa cell complexes are referred as oocytectomized complexes (OOXs) in this study. Fully grown, meiotically competent oocytes were isolated by gentle pipetting of cumulus-oocyte complexes (COCs). Growing, meiotically incompetent oocytes were collected from the preantral follicles of 12-day-old mice by collagenase digestion as described previously [12, 13].
The culture medium used was bicarbonate-buffered MEM
(Life Technologies, Inc., Grand Island, NY) with Earles salts, supplemented with 75 mg/L penicillin G, 50 mg/L streptomycin sulfate, 0.23 mM pyruvate, and 3 mg/ml crystallized lyophilized BSA. To maintain oocytes competent to undergo germinal vesicle breakdown (GVB) at the GV stage, the phosphodiesterase inhibitor milrinone (10 µM) was added to all culture medium regardless of whether oocytes were present or whether the oocytes were incompetent to undergo spontaneous maturation. All medium components were purchased from Sigma Chemical Company (St. Louis, MO). In some experiments, granulosa cells were cocultured with fully grown oocytes or growing oocytes at the concentration of either 2 oocytes/µl or 4 oocytes/µl of culture medium, respectively. These relative concentrations were based on comparable oocyte volume [14]. All cultures were performed in 48-well cell culture plates (#3548, Corning Incorporated, Corning, NY) in 100–150 µl volume of culture medium for 15 h and were maintained at 37°C in a modular incubation chamber (Billups Rothenberg, Del Mar, CA) infused with 5% O2, 5% CO2, and 90% N2.
RNase Protection Assays
Messenger RNA was isolated as described previously [15] from samples of granulosa cells obtained after the various culture experiments presented here. Protected RNA fragments were analyzed and quantified using a Fuji PhosphorImaging System (Fuji Medical Systems USA, Stamford, CT). Data are expressed as the relative steady-state level of Slc38a3 mRNA normalized with mRNA encoded by the housekeeping gene Rpl19. Results are presented as the mean ± SEM of at least three independent experiments.
Uptake of Radiolabeled Amino Acids
To assess the uptake of amino acids, either intact oocyte-cumulus cell complexes, OOX cumulus cells, OOX cumulus cells cocultured with oocytes, or cumulus cell-denuded oocytes were collected after 15 h culture as described previously. They were washed through three dishes of Whitten medium containing 3 mg/ml BSA and 10 µM milrinone and transferred to 150 µl of same medium in 48-well tissue culture plates. Importantly, Whitten medium does not contain free amino acids [16]. The cells were incubated for 3 h in 100 µCi/ml medium containing one of three 3H-labeled amino acids, L-alanine (66 Ci/mmol), L-histidine (50 Ci/ mmole), or L-leucine (300 mCi/mmole) purchased from Dupont NEN (Boston, MA). After incubation, the cells were washed again through three changes of Whitten medium with milrinone and BSA but no labeled amino acids. Incorporation of amino acids into half the intact oocyte-cumulus complexes was determined. Uptake of amino acids into all the other groups is expressed as a percentage of the counts per minute relative to the intact oocyte-cumulus cell complex. The cumulus cells were removed from the other half of the oocyte-cumulus cell complexes, and incorporation of the amino acids into either the oocytes or cumulus cells was measured separately. When incorporation of amino acids into the OOX group that was cocultured with oocytes was measured, the oocytes were removed before assessing uptake into the OOX cumulus cells. Ten oocytes, complexes, or the cumulus cells from 10 complexes were assessed for incorporation of radiolabeled amino acids as described previously [17]. Briefly, the washed cells in 5.0 µl of medium from the final wash dish were transferred to scintillation vials. Two samples were assessed for each group in each experiment, and the experiments were conducted three times independently. For each experiment, a duplicate set of medium blanks (5.0 µl of medium only from the final wash dish) was also prepared. One hundred microliters of 0.1 N NaOH were added to each vial, and samples were held at room temperature for 1 h to dissolve the cells. Then, 0.1 N HCl was added to neutralize the NaOH. The radioactivity in each sample was determined by scintillation spectroscopy. The counts per minute of the blank samples were subtracted from experimental samples.
Statistical Analyses
All experiments were performed at least three times. The Student t-test was used for paired comparisons. A P-value of < 0.05 was considered statistically significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The site(s) of Slc38a3 mRNA expression in the mouse ovary was assessed using in situ hybridization. No expression was detected in the preantral follicles of 12-day-old mice (not shown). Expression of Slc38a3 mRNA in 22-day-old eCG-primed mice was low or undetectable in primordial, primary, or early secondary follicles or in oocytes at any stage of follicular development. However, expression increased in the later stages of secondary (preantral) follicles and was highest in all the granulosa cells, both cumulus and mural, of early tertiary (antral) follicles (Fig. 1). With the progression of antral follicle development, expression diminished to undetectable levels in MGCs but remained elevated in cumulus cells (Fig. 1). This verified the outcome of subtraction hybridization, indicating that the steady-state levels of Slc38a3 mRNA in cumulus cells were higher than in MGCs.
|
Effect of Oocytes on Steady-State Slc38a3 mRNA Levels in Cumulus Cells
To determine whether oocytes of antral follicles affect the steady-state level of Slc38a3 mRNA expressed in cumulus cells, oocytes were microsurgically removed from COCs (OOX), and intact COCs and OOX cumulus cells were cultured for 15 h before assessing relative levels of Slc38a3 transcripts expressed in the cumulus cells using RNase protection assays. In addition, OOX cumulus cells were cocultured with cumulus cell-free FGOs (2 oocytes/ µl) isolated from large antral follicles. OOX resulted in a reduction (P < 0.05) in the steady-state Slc38a3 mRNA to almost undetectable levels in cumulus cells, but coculture of OOX cumulus cells with FGOs restored levels to about the same as seen in the cumulus cells of intact COCs (Fig. 2A). Thus, FGOs promote the expression of Slc38a3 mRNA by cumulus cells.
|
To determine whether the ability of the oocyte to promote Slc38a3 expression by cumulus cells is dependent on the stage of oocyte development, OOX cumulus cells were cocultured with either FGOs (2/µl) or growing oocytes (GOs) (4/µl) isolated from the preantral follicles of 12-day-old mice, and relative steady-state levels of Slc38a3 transcripts present in cumulus cells were determined. The concentration of GO in the cocultures was 4/µl to compensate for an approximate 50% difference in the volume of GOs versus FGOs [14]. FGOs promoted about a 5-fold elevation of Slc38a3 mRNA levels in the cumulus cells, but GOs did not (Fig. 2B). Thus, the ability of oocytes to promote elevated Slc38a3 steady-state mRNA levels in cumulus cells is developmentally regulated.
Little or no expression of Slc38a3 mRNA was detected in the preantral granulosa cells (PAGCs) of 12-day-old mouse ovaries by in situ hybridization (not shown). This raises the question of whether the PAGCs are able to respond to factors from oocytes and elevate steady-state levels of Slc38a3 transcripts. Therefore, OOX cumulus cells (from antral follicles) or OOX PAGCs were cultured either alone or with FGOs for 15 h before assay. Consistent with the results presented previously, FGOs promoted a 7-fold elevation of Slc38a3 mRNA levels by OOX cumulus cells. FGOs also stimulated an elevated level of Slc38a3 mRNA expression in OOX PAGCs (Fig. 2C). Thus, the expression of Slc38a3 mRNA by granulosa cells during follicular development is probably up-regulated by oocytes during their final growth phase, which coincides with their development in late preantral and antral follicles.
Effect of Oocytes on Amino Acid Transport by Granulosa Cells
When Slc38a3 mRNA was injected into Xenopus oocytes, uptake of L-alanine into the frog oocytes was increased 10-fold [9]. This transporter also exhibits a substrate preference for L-histidine and L-glutamine [9]. Therefore, the effect of oocytes on the incorporation of L-alanine and L-histidine into cumulus cells or PAGCs and oocytes was determined. Since transporters in this family do not exhibit a preference for L-leucine [9, 18], the incorporation of this amino acid was also assessed for comparison.
Approximately 15–20% of the L-alanine, L-histidine, and L-leucine incorporated into the intact COC was incorporated into the oocyte, the remainder into the cumulus cells (Fig. 3). However, while about the same amount of L-leucine was incorporated into cumulus cell-denuded oocytes, significantly less L-alanine or L-histidine was incorporated into denuded oocytes compared to cumulus cell-enclosed oocytes (Fig. 3A). These data for L-alanine and L-leucine are consistent with those presented by Colonna and Mangia [7]; however, incorporation of L-histidine was not included in their study. The incorporation of L-alanine and L-histidine but not L-leucine into the oocyte is dependent, either completely or partially, on the participation of cumulus cells and is probably mediated by the gap junctions that metabolically couple oocytes and granulosa cells [8]. Incorporation of all three amino acids into OOX cumulus cells was much less than into the cumulus cells of intact complexes (Fig. 3B). Coculture of OOX cumulus cells with denuded FGOs (2 oocytes/µl) dramatically stimulated incorporation of L-alanine and L-histidine by cumulus cells. For example, about 70% of the total incorporation of L-alanine into the intact COCs was incorporated into the cumulus cells (Fig. 3B). However, incorporation into OOX cumulus cells was only about 3% of the total L-alanine incorporation found in the intact COCs, but incubation of OOX cumulus cells with FGOs increased incorporation to 44% (Fig. 3B). Similar results were obtained when the incorporation of L-histidine was measured (Fig. 3B). FGOs also stimulated incorporation of L-leucine by OOX cumulus cells but not to nearly the same extent as they did for L-alanine and L-histidine (Fig. 3B). Incubation of OOX cumulus cells with FGOs increased L-leucine incorporation from 12% to 20% of the total incorporation into COCs (Fig. 3B). FGOs did not promote the incorporation of any amino acid into OOX cumulus cells to the same extent measured in the cumulus cells of intact complexes, even when the concentration of oocytes used in the coculture was increased to 4/µl (not shown).
|
As shown previously, expression of Slc38a3 mRNA in preantral follicles was either undetectable or only slightly above background. Nevertheless, GOs from preantral follicles might secrete factors that promote amino acid uptake, but PAGCs could be unresponsive to them. Therefore, experiments were conducted to determine whether GOs secrete factors that can promote the incorporation of amino acids by OOX-cumulus cells (from antral follicles), which were shown to be responsive to the stimulatory factors from FGOs. The concentration of GO in the cocultures was 4/µl to compensate for an approximate 50% difference in the volume of GOs versus FGOs [14]. As expected, FGOs increased the OOX cumulus cell incorporation of L-alanine by 11.5-fold and L-leucine by 2.3-fold. However, GOs did not promote the incorporation of either amino acid by OOX cumulus cells (Fig. 4A).
|
The relatively low level of expression of Slc38a3 mRNA by PAGCs could be due either to the failure of GOs to promote expression, as shown previously, or to the inability of PAGCs to respond to the stimulus or both. FGOs not only were able stimulate elevation in the steady-state levels of Slc38a3 mRNA (Fig. 2) but also promoted a 40-fold increase in the incorporation of L-alanine into OOX-PAGCs (Fig. 4B). Nevertheless, FGOs did not promote the uptake of L-leucine into OOX-PAGCs (Fig. 4B).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
SLC38A3 is a sodium-coupled neutral amino acid transporter exhibiting a preference for L-glutamate, L-histidine, and L-alanine but not for L-leucine [9]. In the absence of FGOs, cumulus cell incorporation of L-alanine, L-histidine, and L-leucine was greatly reduced. FGOs cocultured with OOX cumulus cells greatly stimulated the incorporation of L-alanine and L-histidine but stimulated the uptake of L-leucine to a much lesser extent. FGOs were unable to restore the incorporation of any of the amino acids to levels measured in the intact COC even though the steady-state levels of Slc38a3 mRNA were elevated to levels similar to that of the intact COC. Thus, paracrine factors from FGOs stimulate cumulus cell incorporation of amino acids, particularly the amino acids categorized as coupling dependent, and are taken up poorly by the oocyte itself. Maximal incorporation of amino acids by cumulus cells appears to require contact with the oocyte. The most extreme example of this is the uptake of L-leucine by cumulus cells. Expression of Amh mRNA by granulosa cells also appears largely dependent on contact or very close association with the oocyte, particularly in PAGCs, but also in cumulus cells [19]. This apparent requirement for contact between oocytes and granulosa cells for maximum expression of Amh mRNA or for amino acid uptake by the granulosa cells may be dependent on gap junction-mediated signaling. Such signals originating in the granulosa cells may promote the secretion of paracrine factors by the oocyte that, in turn, enhance granulosa cell function in a regulatory loop.
Although it is tempting to assume that the uptake of L-alanine and L-histidine into cumulus cells depends on expression of Slc38a3 mRNA, there is no direct evidence presented here that these are linked. Considering the many transporters involved in amino acid uptake, it is likely that SLC38A3 is only one of several transporters involved in the uptake of these amino acids by cumulus cells. In fact, studies in progress in our laboratory suggest that cumulus cells also express the transporter SLC38A5, and this transporter appears to have a similar substrate preference to SLC38A3 in rats [20].
In addition to Slc38a3, the expression of six genes (Aldoa, Eno1, Ldh1, Pfkp, Pkm2, and Tpi1), encoding enzymes participating in the glycolysis pathway, is greater in cumulus cells than MGCs [6]. It is well documented from classical studies that mouse oocytes, as well as preimplantation embryos, are deficient in their ability to metabolize glucose and that the products of glycolysis provided by the cumulus cells are essential for oocyte development [3–5]. The steady-state levels of all these transcripts encoding glycolytic enzymes are elevated by paracrine factors produced by FGOs but not GOs. Thus, before the LH surge, oocytes probably promote the expression of genes, including Slc38a3 and genes encoding glycolysis enzymes, in cumulus cells that, in turn, are beneficial for oocyte development. Although the role of such gene products in oocyte development is not always obvious, promoting expression of genes involved in amino acid transport, particularly the transport of coupling-dependent amino acids, seems evident. In fact, FGOs promote the incorporation of the coupling-dependent amino acids L-alanine and L-histidine to a significantly greater extent than the coupling independent amino acid L-leucine. The increase in oocyte volume is the greatest in the later stages of preantral follicle and early antral follicle development [13], and the demand for amino acids to support oocyte growth must be greatest at this time. Since oocytes are deficient in their ability to transport some amino acids, such as L-alanine and L-histidine, they probably become increasingly dependent on the cumulus cells to provide them. In effect, oocytes may use the cumulus cell membrane as specialized extension of the oolemma and the coupling gap junctions as means of conveyance. The studies reported here show that oocytes enhance incorporation of crucial amino acids via signals using both paracrine factors and contact-mediated communication. At least part of the enhancement mechanism involves up-regulation of cumulus cell transcripts, such as Slc38a3 and those from genes encoding glycolytic enzymes.
Oocytes regulate the rate of ovarian follicular development [21]. This is most dramatically illustrated by the fact that reaggregation of partly grown oocytes from preantral follicles with the somatic cells of newborn ovaries, which contain only primordial follicles, resulted in a doubling of the rate of follicular development [21]. It is probable that this regulation is mediated by paracrine factors from the oocyte, such as GDF9 and BMP15, and by signals transmitted by contact-mediated mechanisms, such as through gap junctions. Signals originating in the oocyte not only affect the rate of follicular development but also profoundly affect the differentiation of granulosa cells. This is evidenced by the oocyte's influence on different patterns of gene expression among the granulosa cells at different times and in the various populations of granulosa cells. Fully grown oocytes promote the untimely expression of Slc38a3 mRNA and greatly enhance the uptake of coupling-dependent amino acids by preantral granulosa cells. This is an example of how oocytes can drive the precocious development of granulosa cells to ensure the coordination of oocyte and granulosa cell development. Similarly, the ability of oocytes to control energy metabolism by granulosa cells is another mechanism that could regulate the rate of follicular development [6].
The developmental coordination between the oocyte and the somatic components of the follicle ensures that both are at appropriate stages of maturation. Thus, a developmentally competent oocyte is available to be ovulated at the same time that the follicle is competent to undertake the ovulatory process in response to LH. In fact, the oocyte itself plays a key role in the ovulatory process since GDF9 produced by the oocyte is required to promote the production of hyaluronic acid and other factors by cumulus cells that are necessary for ovulation [1, 2]. We propose that oocytes promote the expression of genes involved in cellular metabolism, such as Slc38a3 and the genes encoding glycolysis enzymes [6], to achieve two goals. First, some of these gene products are not produced by the oocyte itself, and promoting the expression of these genes in cumulus cells enhances aspects of metabolic cooperation between the granulosa cells and the oocyte that are essential for the development of the oocyte. Second, the oocytes use their ability to regulate metabolic pathways in the granulosa cells to orchestrate the rate of follicular development.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence. FAX: 207 288 6073; jje{at}jax.org
Received: 9 March 2005.
First decision: 26 March 2005.
Accepted: 18 April 2005.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  C. X. Yeo, R. B. Gilchrist, and M. Lane Disruption of Bidirectional Oocyte-Cumulus Paracrine Signaling During In Vitro Maturation Reduces Subsequent Mouse Oocyte Developmental Competence Biol Reprod, May 1, 2009; 80(5): 1072 - 1080. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. Ratchford, C. R. Esguerra, and K. H. Moley Decreased Oocyte-Granulosa Cell Gap Junction Communication and Connexin Expression in a Type 1 Diabetic Mouse Model Mol. Endocrinol., December 1, 2008; 22(12): 2643 - 2654. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Bettegowda, O. V. Patel, K.-B. Lee, K.-E. Park, M. Salem, J. Yao, J. J. Ireland, and G. W. Smith Identification of Novel Bovine Cumulus Cell Molecular Markers Predictive of Oocyte Competence: Functional and Diagnostic Implications Biol Reprod, August 1, 2008; 79(2): 301 - 309. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. B. Gilchrist, M. Lane, and J. G. Thompson Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality Hum. Reprod. Update, March 1, 2008; 14(2): 159 - 177. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. X. Yeo, R. B. Gilchrist, J. G. Thompson, and M. Lane Exogenous growth differentiation factor 9 in oocyte maturation media enhances subsequent embryo development and fetal viability in mice Hum. Reprod., January 1, 2008; 23(1): 67 - 73. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-Q. Su, K. Sugiura, K. Wigglesworth, M. J. O'Brien, J. P. Affourtit, S. A. Pangas, M. M. Matzuk, and J. J. Eppig Oocyte regulation of metabolic cooperativity between mouse cumulus cells and oocytes: BMP15 and GDF9 control cholesterol biosynthesis in cumulus cells Development, January 1, 2008; 135(1): 111 - 121. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. J Diaz, K. Sugiura, and J. J Eppig Regulation of Pcsk6 Expression During the Preantral to Antral Follicle Transition in Mice: Opposing Roles of FSH and Oocytes Biol Reprod, January 1, 2008; 78(1): 176 - 183. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Y. Li, D. Colley, K. J. Barr, S.-P. Yee, and G. M. Kidder Rescue of oogenesis in Cx37-null mutant mice by oocyte-specific replacement with Cx43 J. Cell Sci., December 1, 2007; 120(23): 4117 - 4125. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Feuerstein, V. Cadoret, R. Dalbies-Tran, F. Guerif, R. Bidault, and D. Royere Gene expression in human cumulus cells: one approach to oocyte competence Hum. Reprod., December 1, 2007; 22(12): 3069 - 3077. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. E Harris, I. Adriaens, H. J Leese, R. G Gosden, and H. M Picton Carbohydrate metabolism by murine ovarian follicles and oocytes grown in vitro Reproduction, September 1, 2007; 134(3): 415 - 424. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. L. Russell and R. L. Robker Molecular mechanisms of ovulation: co-ordination through the cumulus complex Hum. Reprod. Update, May 1, 2007; 13(3): 289 - 312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. J. Diaz, K. Wigglesworth, and J. J. Eppig Oocytes determine cumulus cell lineage in mouse ovarian follicles J. Cell Sci., April 15, 2007; 120(8): 1330 - 1340. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ebner, M. Moser, M. Sommergruber, O. Shebl, and G. Tews Incomplete denudation of oocytes prior to ICSI enhances embryo quality and blastocyst development Hum. Reprod., November 1, 2006; 21(11): 2972 - 2977. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. J. Schweigert, B. Gericke, W. Wolfram, U. Kaisers, and J. W. Dudenhausen Peptide and protein profiles in serum and follicular fluid of women undergoing IVF Hum. Reprod., November 1, 2006; 21(11): 2960 - 2968. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Xing, R. Gosden, P. Lasko, and H. Clarke Murine homologues of the Drosophila gustavus gene are expressed in ovarian granulosa cells. Reproduction, May 1, 2006; 131(5): 905 - 915. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. FitzHarris and J. M. Baltz Granulosa cells regulate intracellular pH of the murine growing oocyte via gap junctions: development of independent homeostasis during oocyte growth Development, February 15, 2006; 133(4): 591 - 599. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
