Localization and synthesis of hyaluronic acid in the cumulus cells and mural granulosa cells of the preovulatory follicle.

Mural and cumulus granulosa cells synthesize hyaluronic acid (HA) and expand in vitro in response to follicle-stimulating hormone and a soluble factor(s) produced by fully grown oocytes. In the present study we examined HA synthesis and extracellular matrix organization by the two cell populations in vivo during the preovulatory period. After injection of human chorionic gonadotropin into pregnant mares' serum gonadotropin-primed animals, a progressive increase in HA synthesis was observed by the cumulus cell-oocyte complex (COC), and by the mural granulosa cells adjacent to the antrum (antral granulosa cells). The outermost layers of mural granulosa cells (peripheral granulosa cells) did not synthesize HA. Net HA synthesis was approximately 4 pg/cell for COCs isolated after full expansion induced either in vivo or in vitro, whereas the total HA content and cell number in the ovulated COC (approximately 11 ng HA and approximately 3000 cells per COC) were about threefold higher than for COCs expanded in vitro (approximately 4 ng HA and approximately 1000 cells per COC). The increased cell content of ovulated COCs appears to be primarily the result of inclusion of proximal mural granulosa cells which synthesize HA in response to the oocyte factor(s) and become incorporated in the expanded COC extracellular matrix mass. Media conditioned by oocytes enclosed in the cumulus cell mass (intact COCs) contained only 10-20% of the HA-stimulatory activity of media conditioned by an equal number of isolated oocytes when tested on mural granulosa cell cultures. Further, HA-stimulatory activity of media conditioned by isolated oocytes was dramatically reduced (approximately 70%) by preincubation for 5 hr with cumulus cells compared to preincubation in the absence of cells. The results suggest that differences in HA synthesis between subregions of membrana granulosa depend on a diffusion gradient of the oocyte factor(s).     

Qualitative and quantitative changes in protein synthesis of bovine follicular cells during the preovulatory period.

To understand the mechanisms governing oocyte maturation better, the effects of the gonadotropin surge were studied on follicular cells of bovine preovulatory follicles. For this purpose, qualitative and quantitative changes in protein synthesis by both granulosa cells and cumulus cells were compared relative to the luteinizing hormone (LH) surge and the resumption of meiosis in the oocyte. Follicular cells were collected at different times before and up to 25 hr after the LH surge. For each individual preovulatory follicle, granulosa and cumulus cells were incubated separately for 3 hr with 3H-methionine or with 35S-methionine. Newly synthesized cytosolic proteins from granulosa and cumulus cells and proteins secreted into the medium were analyzed by polyacrylamide gel electrophoresis. The radioactivity was measured by liquid scintillation counting after slicing of the gels or revealed by fluorography. Three major peaks of the newly synthesized proteins, with molecular weights of 76, 56, and 30 kDa, were studied throughout the preovulatory period. After the LH surge, the overall level of protein synthesis increased in granulosa cells. In addition, the pattern of cytosolic proteins in granulosa cells changed, and, in particular, the relative synthesis of the 30 kDa peak decreased. These changes in cytosolic protein synthesis may be due to the action of LH since they could be reproduced in vitro in LH-stimulated granulosa cells. A predominant peak of 56 kDa was secreted by granulosa cells throughout the experimental period. No significant change was observed in proteins synthesized by cumulus cells under the same experimental conditions. The amounts of radioactivity incorporated into the three major proteins secreted by granulosa cells, however, were correlated significantly with the amounts of radioactivity incorporated by similar proteins synthesized by cumulus cells. These results indicate that cumulus cells respond differently from granulosa cells to the gonadotropin surge but not in an independent manner.     

Gene expression profiles of cumulus cell oocyte complexes during ovulation reveal cumulus cells express neuronal and immune-related genes: does this expand their role in the ovulation process?

Ovulation is a complex process initiated by the preovulatory LH surge, characterized by cumulus oocyte complex (COC) expansion and completed by the release of a mature oocyte. Although many ovarian genes that impact ovulation have been identified, we hypothesized that genes selectively expressed in COCs would be overlooked by approaches using whole ovary or granulosa cell samples. RNA isolated from COCs collected from preovulatory follicles of equine chorionic gonadotropin (CG) primed mice and at selected times after human CG treatment was subjected to microarray analyses and results confirmed by RT-PCR analyses, Western blotting, and immunofluorescent studies. A remarkable number of genes were up-regulated in COCs including Areg, Ereg, and Btc. Several genes selectively expressed in cumulus cells compared with granulosa cells were related to neuronal (Mbp, Tnc, Nts) or immune (Alcam, Pdcd1, Cd34, Cd52, and Cxcr4) cell function. In addition to Sfrp2, other members of the Wnt/Fzd family (Sfrp4, Fdz1 and Fdz2) were expressed in COCs. Thus, there is a cumulus cell-specific, terminal differentiation process. Furthermore, immunofluorescent analyses documented that cumulus cells are highly mitotic for 4-8 h after human CG and then cease dividing in association with reduced levels of Ccnd2 mRNA. Other down-regulated genes included: Cyp19a1, Fshr, Inhb, and the oocyte factors Zp1-3 and Gja4. In summary, the vast number of matrix, neuronal, and especially immune cell-related genes identified by the gene- profiling data of COCs constitutes strong and novel evidence that cumulus cells possess a repertoire of immune functions that could be far greater than simply mediating an inflammatory-like response.     

Transient invasive migration in mouse cumulus oocyte complexes induced at ovulation by luteinizing hormone

Ovulation, the release of the oocyte from the ovarian follicle, is initiated by the luteinizing hormone surge. It is clear that highly controlled degradation of the follicle and ovarian wall is required for passage of the oocyte and accompanying cumulus cells from the follicle, but the mechanism has not yet been elucidated. Here we show that cumulus oocyte complexes (COCs) adopt transient adhesive, migratory, and matrix-invading capacities at the time of ovulation. We characterized cell adhesion, migration, and invasion in preovulatory and postovulatory mouse COCs collected over a time course post-human chorionic gonadotropin (hCG) administration. Adhesion of dispersed cumulus cells and intact COCs to extracellular matrix proteins present in the ovarian wall (collagens, laminin, and fibronectin) increased significantly after hCG treatment and declined immediately after ovulation. Cumulus cell migration was low in unexpanded, equine chorionic gonadotropin-only treated COCs, but increased 4, 8, and 10 h post-hCG, reaching a peak at 12 h post-hCG that coincided with ovulation. The ability of cumulus cells to migrate was rapidly diminished in COCs isolated from the oviduct within 2 h postovulation. Cell migration was cumulus cell specific and was not observed in granulosa cells. Invasion through three-dimensional collagen I and matrigel barriers by preovulatory expanded COCs was equivalent to that of a known invasive breast cancer cell line (MB-231). Cumulatively, these results demonstrate that cumulus cells in the expanded COC transition to an adhesive, motile, and invasive phenotype in the periovulatory period that may be required for successful release of the oocyte from the ovary at ovulation.     

Secretion of cumulus expansion-enabling factor (CEEF) in porcine follicles

The objective of this study was to find out whether porcine cumulus and mural granulosa cells can secrete cumulus expansion-enabling factor (CEEF). Culture drops of M-199 medium were conditioned with denuded porcine oocytes (1 oocyte/μl), cumulus cells from oocytectomized complexes (1 OOX/μl), pieces of mural granulosa isolated from preantral to preovulatory follicles (1000 cells/μl), or oviductal cells (1000 cells/μl) for 24 hr. The production of CEEF was assessed by the addition of mouse OOX and follicle-stimulating hormone (FSH) (1 μg/ml) to microdrops of the conditioned medium. After 16–18 hr, expansion of the mouse OOX was scored on a scale of 0 to 4 by morphologic criteria. Mouse OOX did not expand in nonconditioned FSH-supplemented medium. Immature porcine oocytes produced +3 to +4 expansion of the mouse OOX. Granulosa cells isolated from preantral and early antral follicles and cumulus cells isolated from all stages of follicle development constitutively secreted CEEF under in vitro conditions. Mural granulosa cells of small, medium, and preovulatory (PMSG) follicles also secreted CEEF in vitro; however, FSH or leutenizing hormone (LH) stimulation was essential for this secretion. Hormonally induced secretion of CEEF was accompanied by expansion of the mural granulosa itself. Granulosa cells isolated from follicles of gilts 20 hr after PMSG and human chorionic gonadotropin (hCG) administration did not produce CEEF and did not expand in response to FSH and LH in vitro. CEEF activity also was found in the follicular fluid of small antral follicles, was reduced in medium follicles, and was not detectable in PMSG-stimulated follicles. However, CEEF activity was reestablished in the follicular fluid of preovulatory follicles by hCG injection, conceivably due to increased production of CEEF by cumulus cells. We conclude that (1) porcine cumulus and mural granulosa cells are capable of CEEF production in vitro and (2) autocrine secretion of CEEF by cumulus cells is involved in regulation of porcine cumulus expansion both in vitro and in vivo. Mol. Reprod. Dev. 49:141–149, 1998. © 1998 Wiley-Liss, Inc.     

Apoptosis within bovine follicular cells and its effect on oocyte development during in vitro maturation

Developmental competence of oocytes is compromised if they originate from atretic follicles. Apoptosis is the underlying process of atresia. Apoptotic changes in follicular cells are thought to influence the outcome of IVF. The aim of this study was to investigate apoptosis in different compartments of single bovine follicles (follicular wall, granulosa and cumulus cells (CC)) in relation to COC morphology, and to determine whether the addition, in vitro, of exogenous follicular cells from atretic follicles to maturing cumulus oocyte complexes (COCs) influenced the development of oocytes.Antral follicles were dissected from bovine ovaries and opened to obtain COCs and free floating granulosa cells (GC). The COCs were classified according to morphology. Apoptosis was determined in cumulus and granulosa cells and in homogenates of the remaining follicular wall.For every morphological class of COCs, a large variability of apoptotic expression was found in all follicle compartments. Follicular wall apoptosis was not correlated to COC morphology or to the percentage of apoptotic granulosa or cumulus cells. In grade 1 (best morphology) COCs, the degree of apoptosis in granulosa cells was comparable to cumulus cell apoptosis (P<0.01). The overall expression of apoptosis in granulosa cells of follicles containing grade 3 COCs (median+/-median absolute deviation: 37.8+/-13.8%) was significantly higher (P<0.05) than in follicles with grade 1 (22.7+/-10.4%) or grade 2 COCs (20.0+/-17.0%). About 48.3% of grade 3 COCs possessed strongly apoptotic cumulus cells compared to 27.8 and 28.2% of grade 1 or grade 2 COCs, respectively. Nonapoptotic cumulus complexes were observed in grades 1 and 2 COCs only.Adding exogenous follicular cells from atretic follicles to bovine COCs (grades 1 and 2) during in vitro maturation (IVM) had no impact on fertilization, blastocyst formation or hatching after IVF. This is of particular practical relevance to embryo production after ovum pick up (OPU), as during this process, good quality COCs are cultured together with simultaneously collected slightly atretic COCs.     

Developmental and hormonal regulation of cumulus expansion and secretion of cumulus expansion-enabling factor (CEEF) in goat follicles

The objective of this article was to study the developmental and hormonal regulation of cumulus expansion and secretion of cumulus expansion-enabling factor (CEEF) in goat follicles. M-199 medium was conditioned for 24 hr with cumulus-denuded oocytes (DOs), oocytectomized complexes (OOXs), or mural granulosa cells (MGCs) from goat follicles of different sizes. Mouse OOXs and eCG were added to culture drops of the conditioned medium and cumulus expansion was scored at 18 hr of culture to assess CEEF production. While mouse OOXs did not expand, goat OOXs underwent full cumulus expansion when cultured in nonconditioned eCG-supplemented M-199 medium. When cultured in nonconditioned medium containing 10% follicular fluid (FF) from goat medium (2-4 mm) and small (0.8-1.5 mm) follicles, 71-83% mouse OOXs expanded; but expansion rates decreased (P < 0.05) at either lower or higher FF concentrations. FF from large (5-6 mm) follicles did not support mouse OOX expansion at any concentrations. While medium conditioned with DOs from follicles of all the three sizes supported expansion of 80-90% mouse OOXs, medium conditioned with mature DOs had no effect. While cumulus cells from follicles of all the three sizes secreted CEEF in the absence of gonadotropins, MGCs from large follicles became gonadotropin dependent for CEEF production. Both FSH and LH stimulated CEEF production by large follicle MGCs, but FSH had a shorter half-life than LH to expand mouse OOXs. Few meiosis-incompetent goat oocytes from small follicles underwent cumulus expansion when cultured in medium conditioned with goat DOs or cocultured with goat COCs from medium follicles. It is concluded that (1) goat cumulus expansion is independent of the oocyte; (2) the limited CEEF activity in FF from large follicles was due mainly to the inability of MGCs in these follicles to secret the factor in absence or short supply of gonadotropins; (3) the cumulus expansion inability of the meiosis incompetent goat oocytes was due to the inability of their cumulus cells to respond to rather than to produce CEEF.     

Characterization of cumulus expansion-inhibiting factor (CEIF) in goat follicles

Studies suggest that oocyte cumulus expansion is regulated by both cumulus expansion-enabling factor (CEEF) and cumulus expansion-inhibiting factors (CEIF). Many reports on CEEF have appeared, but CEIF has rarely been studied. By cumulus expansion assays using mouse cumulus-oocyte complexes (COCs) and oocytectomized complexes, the present study demonstrated that whereas follicular fluid (FF) from medium (diameter, 2-4 mm) goat follicles contained both CEEF and CEIF activities, FF from large (diameter, 5-6 mm) abattoir or large (diameter, 5-7 mm) follicle-stimulating hormone (FSH)-stimulated follicles contained neither. FF from (diameter, 5-7 mm) human chorionic gonadotropin-stimulated follicles showed CEEF but not CEIF activity. Whereas medium conditioned with cumulus or mural granulosa cells from medium goat follicles contained only CEEF activity, theca cell-conditioned medium (CM) showed both CEEF and CEIF activities. Whereas 0.01 mg/ml of heparin efficiently inhibited cumulus expansion of mouse COCs in vitro, FF from large follicles that showed no CEIF activity contained much higher concentrations (0.23-0.25 mg/ml) of heparin. None of the glycosaminoglycans (GAGs) tested inhibited cumulus expansion of goat COCs. Among the follicles observed, only FF from medium goat follicles contained a linoleic acid (LA) level sufficient to inhibit cumulus expansion of both mouse and goat COCs in vitro. CM contained some amount of GAGs but no LA. Taken together, the results suggest that 1) the FSH and luteinizing hormone (LH) surges before ovulation promote cumulus expansion by down-regulating CEIF and up-regulating CEEF activity, respectively; 2) GAGs are not the CEIF in goat follicles; and 3) LA has CEIF activity but additional factors must be involved, because CM that showed high CEIF activity contained no LA.     

Production of progesterone from de novo-synthesized cholesterol in cumulus cells and its physiological role during meiotic resumption of porcine oocytes

To investigate the role of factors secreted by cumulus cells during meiotic resumption of porcine oocytes, 1, 5, 10, or 20 cumulus-oocyte complexes (COCs) were cultured in each well of a culture dish containing 300 microl of maturation medium for 20 h. There was a significant positive correlation between the rate of germinal vesicle breakdown (GVBD) and the number of COCs cultured in each well for 20 h. The level of progesterone in the medium in which COCs had been cultured for 20 h also rose significantly with an increase in the number of COCs cultured in each well. A significantly small proportion of GVBD in oocytes when one COC was cultured in each well for 20 h was improved by the addition of progesterone. This proportion of GVBD was fully comparable to that of COCs cultured in the absence of additional progesterone with 20 COCs. Thus, progesterone secreted by COCs plays a positive role in GVBD induction in porcine oocytes. Furthermore, we also examined the role of sterol biosynthesis on progesterone production by cumulus cells and in oocyte GVBD. The results showed that the addition of ketoconazole, which suppressed the sterol biosynthetic pathway produced by demethylation of lanosterol, decreased the rate of GVBD, as well as progesterone production in COCs cultured for 20 h. However, the suppression of GVBD by ketoconazole was overtaken by the addition of progesterone. These results demonstrate that a high level of progesterone produced by cumulus cells was responsible for an acceleration of GVBD in porcine oocytes.     

Induced expression of pattern recognition receptors in cumulus oocyte complexes: novel evidence for innate immune-like functions during ovulation

Ovulation is the complex, inflammatory-like process by which the cumulus oocyte complex (COC) is released from a mature, preovulatory follicle through a rupture site at the ovarian surface and requires expression of genes that generate and stabilize the expanded extracellular COC matrix. Gene profiling analyses of COCs at selected time intervals during ovulation revealed that many genes associated with immune related surveillance functions were also induced in cumulus cells. Specifically, cell surface signaling molecules known as pattern recognition receptors that act as sensors of the external environment important for the innate immune system to detect self from nonself or altered self are induced and/or expressed in cumulus cells as well as granulosa cells. These include the complement factor q1, CD14, and the Toll-like receptors (TLRs) 4, 8, and 9 as well as mediators of TLR activation, myeloid differentiation primary response gene 88 and interferon regulatory factor 3. COCs exposed to bacterial lipopolysaccharide exhibit enhanced phosphorylation of p38MAPK, ERK1/2 and nuclear factor-kappaB and increased expression of Il6 and Tnfa target genes, documenting that the TLR pathway is functional. Cumulus cells and granulosa cells also express the scavenger receptors CD36 and scavenger receptor type B1 and exhibited phagocytic uptake of fluorescently tagged bacterial particles. Collectively, these results provide novel evidence that cumulus cells as well as granulosa cells express innate immune related genes that may play critical roles in surveillance and cell survival during the ovulation process.     

Development of functional LH Receptors on pig cumulus–oocyte complexes cultured in vitro by a novel two‐step culture system

We show in the present study that freshly isolated pig cumulus–oocyte complexes (COCs) display a limited response to LH, as assessed by the expression of hyaluronan synthase 2 (Has2) mRNA, activation of protein kinase A (PKA), production of hyaluronic acid (HA) and progesterone, cumulus cell expansion and resumption of meiosis. These data indicate that freshly isolated COCs do not possess a sufficient number of functional LH receptors (LHR). However, the expression of Lhr significantly increased during the culture of COCs in vitro in a medium supplemented with FSH. Assuming that the effect of FSH on LHR induction is mediated via cAMP signaling pathways, we developed a new culture system, in which the COCs were pre‐cultured for 72 hr in a medium supplemented with dbcAMP. The pre‐cultured COCs remained in the germinal vesicle stage, their cumulus investment underwent a dramatic increase in size and gap junctions between the cumulus cells were preserved. The stimulation of such COCs with either FSH or LH led to the resumption and completion of meiosis, activation of PKA, expression of Has2, synthesis of large amounts of HA and progesterone, and extensive expansion of cumulus cells. We conclude that the formation of functional LHR is stimulated in cumulus cells during the culture in vitro in a cAMP‐dependent pathway. The dbcAMP‐treated COCs thus represent a new model in which the resumption of meiosis and cumulus expansion can be induced exclusively by the action of recombinant LH. Mol. Reprod. Dev. 76: 751–761, 2009. © 2009 Wiley‐Liss, Inc.     

Evidence of new antigens in the mouse cumulus oophorus during preovulatory cumulus expansion

The effects of an antiserum (anti-COC) against ovulated mouse cumulus-oocyte complexes (COC) on in vitro fertilization of mouse oocytes were studied. Preincubation of ovulated COC with various concentrations of anti-COC led to dose-dependent impairment of fertilization rates as well as to a decrease in the number of spermatozoa attached to the zona pellucida. Anti-COC was used to probe Western blots of cumulus proteins. These cumuli were obtained from 2 experimental groups of mice corresponding to 2 different maturational stages (preovulatory immature COC or preovulatory mature COC). Two antigens (70 and 80 kDa) present in cumulus intercellular matrix from mature COC were only found as traces in matrix from immature COC. In addition, the protein pattern of the cumulus intercellular matrix was different from that of cumulus cells, whatever the COC maturational stage. These results indicate the appearance of new proteins in the cumulus oophorus during preovulatory expansion and are consistent with the contraceptive action of anti-COC. © 1993 Wiley-Liss, Inc.     

Pig membrana granulosa cells prevent resumption of meiosis in cattle oocytes

Membrana granulosa was isolated from healthy large antral follicles of prepubertal or cyclic gilts stimulated with PMSG or PMSG and hCG. Ultrastructural observations revealed that pieces of pig membrana granulosa were associated with the basement membrane. The cattle cumulus-enclosed oocytes (COC) were placed in the rolled pieces of the pig membrana granulosa (PMG). After 8 and 24 hr of coculture with PMG from prepubertal gilts, only 16% and 21% of oocytes underwent GVBD, respectively. PMG from PMSG-stimulated cyclic gilts blocked the resumption of meiosis in all COC. The inhibitory effect of heterologous granulosa cells was fully reversible. When COC were initially incubated for 2 and 4 hr, subsequent culture in PMG prevented GVBD in 100% and 36% of oocytes, respectively. This suggests that functional contact between COC and PMG was established during the first 2 hr of coculture. To follow metabolic cooperation between PMG and COC, PMG was prelabeled with ³H-uridine and cocultured with COC. Autoradiography on semithin sections revealed the intensive passage of ³H-uridine from PMG into the cumulus layer and an oocyte. COC placed in PMG after GVBD (8 and 12 hr of an initial incubation) did not extrude the first polar body. PMG isolated from cyclic gilts after PMSG and hCG stimulation also inhibited GVBD of COC. Since nearly all COC placed in PMG isolated 10 and 12 hr after hCG remained in the GV stage after 24 hr of coculture, the hCG stimulation did not substantially diminish the meiosis inhibiting activity of PMG. During coculture, cattle cumulus cells were closely associated with the basement membrane, but no gap junctions were formed among heterologous granulosa cells. These results suggest that an inhibitory factor secreted by pig granulosa cells is not species specific and it can act in vitro without the mediation of gap junctions. © 1993 Wiley-Liss, Inc.     

Effect of intraovarian factors on porcine follicular cells: cumulus expansion, granulosa and cumulus cell progesterone production

The role of granulosa cell conditioned media (CM) containing luteinization stimulator (LS), and the role of EGF in the cumulus expansion of oocyte-cumulus complexes (OCC) isolated from large antral follicles was investigated. The CM were prepared by incubation of granulosa cells isolated from large antral follicles. After 24h incubation, more than 61 or 64% of OCC expanded to the +3 and +4 stage in the presence of CM (50%) or EGF (10ng/ml), respectively. The stimulatory effect of LS and EGF on the cumulus expansion was accompanied by the enhanced hyaluronic acid synthesis. Complete suppression of cumulus expansion stimulated by LS and EGF was observed in the presence of 10 micromol/l genistein (tyrosine kinase inhibitor), in the presence of 10mmol/l LiCl (the inhibitor of inositol 1,4,5-trisphosphate metabolism), and 100 micromol/l gallopamil, verapamil and norverapamil (calcium channel blockers). Stimulatory effect of EGF on the cumulus expansion of OCC isolated from large follicles was accompanied by the increased cumulus cell progesterone production. However, EGF did not affect the progesterone production by OCC isolated from small follicles. To determine whether EGF could modulate the granulosa cell steroidogenesis also, the effect of EGF on granulosa cells isolated from large (LGC) and small (SGC) follicles was compared. EGF (10ng/ml) failed to affect the progesterone synthesis during 72h culture of SGC but significantly enhanced the LGC progesterone production. Our results indicate that luteinization factor stimulates the cumulus expansion and hyaluronic acid synthesis by the OCC isolated from large antral follicles. The mechanism of LS- and EGF-induced cumulus expansion may involve tyrosine kinase activation and calcium mobilization. In addition, these results indicate the different response of porcine cumulus and granulosa cells originating from small and large follicles on the stimulatory effect of EGF.