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.     

The potential role of gap junction communication between cumulus cells and bovine oocytes during in vitro maturation

Preliminary studies in our laboratory have indicated that modulating cumulus expansion early or late during culture has a profound influence on the subsequent development of cumulus-enclosed oocytes. Our objectives were to evaluate the effect of short term exposure to recombinant human follicle-stimulating hormone (r-hFSH) during in vitro maturation (IVM) on cumulus expansion and developmental competence of bovine oocytes. A highly significant (P < 0.0001) improvement in blastocyst development rate as a proportion of cleaved oocytes after IVM of oocytes was observed in the presence of r-hFSH for the first 6 hr of culture. To demonstrate the importance of the functional coupling between the oocyte and the cumulus compartment during that period of 6 hr, cumulus-oocyte complexes (COCs) were matured with r-hFSH for the first 6 hr followed by 18 hr in presence of 1-heptanol or 1-octanol (gap junction inhibitors) to block the communication between the two. With the coupling inhibitors, the blastocyst yield was significantly decreased (P < 0.05). A brief treatment (30 min) with the weak base methylamine, known to reverse the gap junction inhibitors effect, significantly (P < 0.05) reversed the inhibitory action of these agents on the blastocyst rate. Gap junction communication between the oocyte and surrounding cumulus cells was further studied using microinjection of the fluorescent dye Lucifer Yellow. Morphological evidences (dye transfer) were obtained that support the presence of functional coupling for a longer period with the FSH short exposure. In conclusion, high developmental rates of bovine oocytes can be achieved with a short exposure to r-hFSH. This effect is believed to be mediated through gap junctions as developmental competence of oocytes is compromised by the inhibition of their function.     

Hypoxanthine (HX) inhibition of in vitro meiotic resumption in goat oocytes

To improve in vitro maturation and to understand the mechanism for meiotic resumption of oocytes, meiotic progression, and its control by hypoxanthine (HX) were studied in goat oocytes. Ovaries were obtained from a local abattoir, and cumulus-oocyte complexes (COCs) and follicular fluid were collected from follicles of different surface diameters (SDs). The meiotic competence and progression of oocytes were observed, and the concentration of HX in the follicular fluid and culture media was measured by high-performance liquid chromatography (HPLC). Full meiotic competence of goat oocytes was acquired in follicles of >/=1.5 mm in SD with 90% of the oocytes developing to metaphase II (MII) stage after 24 hr in culture. The HX concentration in follicular fluid decreased with follicle development, from the highest level of 1.16 mM in /=5 mm follicles. HX inhibited meiotic resumption of goat oocytes in a concentration-related manner but this inhibitory effect declined gradually. When we renewed the medium at 4 hr of HX-199 (TCM-199 supplemented with 4 mM HX) culture, the percentage of oocytes with intact germinal vesicle (GV) did not increase but decreased significantly instead. HPLC measurement of HX in the HX-199 culture drops indicated that the HX concentration declined from 0 hr to 4 hr of culture and after medium renewal at 4 hr of culture. By adding dibutyryl cAMP (db-cAMP) at medium renewal, we found that db-cAMP held up the decline of GV percentages. Together, these results were consistent with the possibility that the decline of HX inhibitory effect was not due to HX depletion but rather due to the negative feedback of the metabolites on its further uptake by oocytes. Goat oocytes were capable of normal nuclear maturation and activation after temporal arrest by HX, but prolonged exposure to HX induced spontaneous activation.     

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.     

Factors affecting the efficiency and reversibility of roscovitine (ROS) block on the meiotic resumption of goat oocytes

Goat oocytes from 2 to 4 and 0.8 to 1.2-mm follicles were freed (DOs) or not (COCs) of cumulus cells and cultured for different times in an inhibition medium supplemented with different concentrations of roscovitine (ROS). At the end of culture, oocytes were either cultured in a maturation medium for 24 hr and activated chemically for embryo development, or examined for GV chromatin configurations. Nuclear status was checked at different time points during maturation culture. Although both 200 and 250 microM ROS maintained 78-85% of oocytes at the GV stage for 24 hr, only oocytes blocked with 200 microM ROS developed to MII stage at a high rate after maturation culture. While few oocytes blocked with 200 microM ROS for 24 hr developed into morulae and none into blastocysts after activation, percentages of oocytes developing into morulae and blastocysts increased to the level of the control oocytes when the block time was reduced to 8 hr. While the GV and pMI stages were shortened with MI, and A/TI unaffected after oocytes were blocked for 8 hr, all the stages but A/TI were shortened after 24 hr of block. The sizes of nucleoli diminished with time and the GV chromatin configuration changed during ROS block. Significantly more DOs than COCs were blocked with 200 microM ROS, but none of the blocked DOs matured after drug withdrawal. However, maturation of the DOs improved significantly when ROS concentration was reduced to 150 microM or DOs were co-inhibited with COCs. The GV intact percentages of DOs did not differ after ROS inhibition with or without eCG, but those of COCs decreased significantly after ROS inhibited in the presence of eCG. When MII-incompetent oocytes from 0.8 to 1.2-mm follicles were inhibited with ROS for 8 and 24 hr prior to maturation culture, nuclear maturation improved significantly, activation rates were as high as that of the control oocytes, and some of the activated developed to 4- or 8-cell stages. It is concluded that (i) the efficiency and reversibility of ROS block was both drug concentration and exposure-time dependent; (ii) cumulus cells alleviated the toxicity of ROS on goat oocytes; (iii) eCG released goat oocytes from ROS block through the mediation of cumulus cells; (iv) ROS block quickened the nuclear maturation of goat oocytes and improved the developmental competence of meiosis-incompetent oocytes, possibly due to a sustained nuclear activity during inhibition culture; (v) oocyte nuclear maturation and activation did not depend upon cumulus expansion, but the embryo development occurred in association with cumulus expansion.     

The art and artifact of GDF9 activity: cumulus expansion and the cumulus expansion-enabling factor

The process of cumulus cell expansion is critical for normal fertility. Oocyte-produced growth and differentiation factor 9 (GDF9) has been thought to play a leading role in this process. Recent studies both support and refute this hypothesis. Central to understanding the physiology of GDF9 is the use of recombinant ligand in in vitro assays. There are several laboratories that currently produce recombinant GDF9 preparations that appear to show variable effects on granulosa cell gene expression and cumulus cell expansion. Several of these studies are reviewed here. Standardization in preparation for recombinant GDF9, as well as a more biochemical analysis of the oocyte-secreted forms of GDF9, may help to resolve the conflicts currently seen in the literature.     

EGF-induced EGF-receptor and MAP kinase phosphorylation in goat cumulus cells during in vitro maturation

EGF has been shown to influence meiotic maturation and development competence of oocyte in various mammalian species. We previously reported, in goat, that the EGF receptor (EGF-R) was present both on cumulus cells and oocytes. Here, EGF-induced signaling was investigated during the in vitro maturation process in goat cumulus-oocyte complexes (COCs). Cumulus cells and oocytes were subjected to Western immunoblotting analysis using anti-MAP kinase, anti-phosphotyrosine, anti-phospho MAP kinase, and anti-phospho EGF-R antibodies. We demonstrated that treatment with EGF during the in vitro maturation process induced rapid tyrosine phosphorylation of EGF-R in a time and concentration dependent manner in cumulus cells. A similar pattern of activation by phosphorylation was observed for MAP kinase upon EGF stimulation. AG 1478, an inhibitor of the EGF kinase, suppressed EGF-stimulated phosphorylation of EGF-R and also affected the MAP kinase activation. Treatment with the MEK inhibitor PD 98059 abolished EGF-induced MAP kinase activation. We did not observe oocyte EGF-R phosphorylation in our experiments during the in vitro maturation process. Our data indicate, in goat cumulus cells, that activation of EGF-R by EGF triggers signaling through the MAP kinase pathway during in vitro maturation. This supports the hypothesis that the major site of action for EGF, that regulates oocyte maturation, is the cumulus cell.     

Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation

The expansion, or mucification, of the mouse cumulus oophorus in vitro requires the presence of an enabling factor secreted by the oocyte as well as stimulation with follicle-stimulating hormone (FSH). This study focuses on (1) the ability of mouse oocytes to secrete the enabling factor at various times during oocyte growth and maturation, (2) the temporal relationships between the development of the capacity of the oocyte to undergo germinal vesicle breakdown, the ability of the oocyte to secrete cumulus expansion-enabling factor, and the capacity of the cumulus oophorus to undergo expansion, and (3) the role of the oocyte in the differentiation of granulosa cells as functional cumulus cells. Growing, meiotically incompetent oocytes did not produce detectable amounts of cumulus expansion-enabling factor, but fully grown meiosis-arrested oocytes, maturing oocytes, and metaphase II oocytes did. Detectable quantities of enabling factor were produced by zygotes, but not by two-cell stage to morula embryos. The ability of oocytes to secrete cumulus expansion enabling factor and the capacity of cumulus cells to respond to FSH and the enabling factor are temporally correlated with the acquisition of oocyte competence to undergo germinal vesicle breakdown. Mural granulosa cells of antral follicles do not expand in response to FSH even in the presence of cumulus expansion-enabling factor, showing that mural granulosa cells and cumulus cells are functionally distinct cell types. The perioocytic granulosa cells of preantral follicles isolated from 12-day-old mice differentiate into functional cumulus cells during a 7-day period in culture. Oocytectomized granulosa cell complexes grown in medium conditioned by either growing or fully grown oocytes were comparable in size to intact complexes and maintained their 3-dimensional integrity to a greater degree than oocytectomized complexes grown in unconditioned medium. After 7 days, the oocytectomized complexes were stimulated with FSH in the presence of enabling factor, but no expansion was observed whether or not the oocytectomized complexes grew in the presence of oocyte-conditioned medium. These results suggest that a factor(s) secreted by the oocyte affects granulosa cell proliferation and the structural organization of the follicle, but continual close association with the oocyte appears necessary for the differentiation of granulosa cells into functional cumulus cells, insofar as they are capable of undergoing expansion.     

Developmental potential of oocytes fertilized by conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) after cryopreservation and mesometrial autotransplantation of rabbit ovarian tissue

Objective: To evaluate mesometrial transplantation of frozen-thawed ovarian tissue in rabbit and to choose the optimized fertilization method for oocytes retrieved from grafts by investigating the capability of oocyte fertilization and further development. Forty rabbits were divided into three groups randomly: control group, fresh tissues transplantation group and frozen-thawed tissues transplantation group. Three months after the transplantation, rabbits were stimulated with FSH and oocytes were retrieved 13 h after human chorionic gonadotropin (HCG) injection. Oocytes matured in vivo or in vitro were then fertilized by conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), followed by observation and evaluation of fertilization rate and blastocyst formation rate. Blastocytes embryos were transferred to pseudopregnancy rabbits to observe pregnancy rate and birth rate. There were no significant differences in the percentage of oocytes matured either in vivo or in vitro among the three groups. The fertilization rate, cleavage rate and blastocyst formation rate of in vivo-matured oocytes had no difference among the three groups, whether they were fertilized by IVF or ICSI. Significantly higher fertilization rates of in vitro-matured oocytes were observed with ICSI compared with IVF in each group. The blastocyst formation rate of in vitro-matured oocytes was significantly lower than that of in vivo-matured oocytes in each group. The birth rate of in vivo-matured oocytes was significantly higher than that of in vitro-matured oocytes, although the pregnancy rate was similar between them. Mesometrial transplantation of frozen-thawed ovarian tissue may provide favorable conditions for follicle development. Oocytes retrieved from mesometrial grafts can develop to the blastocyst stage and produce live offspring. ICSI can optimize the fertilization rate of in vitro-matured oocytes retrieved from grafts.     

The mechanism of cumulus cell-oocyte uncoupling: Evidence for the participation of both cumulus cells and oocytes

Cumulus cells are metabolically coupled to oocytes via heterologous gap junctions. This coupling terminates near the time of ovulation, and the termination appears to be correlated with the mucification of the cumulus cells lying immediately adjacent to the oocytes. The first objective of this project was to determine whether follicle stimulating hormone (FSH) induction of cumulus cell-oocyte uncoupling could occur independently of FSH-stimulated cumulus mucification (expansion). Intercellular coupling was measured as a percentage of radiolabeled choline (or its metabolites) that was incorporated into the oocyte relative to the total amount of radiolabel incorporated into the entire cumulus cell-oocyte complex. It was found that the complete suppression of FSH-stimulated cumulus expansion with chondroitin sulfate B had no suppressive effect on FSH-stimulated cumulus cell-oocyte uncoupling. This finding showed that FSH-stimulated cumulus expansion was not required for cumulus cell-oocyte uncoupling. Since 17β-estradiol, testosterone, or progesterone could not induce maximal cumulus cell uncoupling, it was concluded that the uncoupling-promoting action of FSH was probably not mediated by steroid hormones. A partial uncoupling of cumulus cells and oocytes was found when spontaneous oocyte maturation had occurred in the absence of FSH. This partial uncoupling was prevented by incubation of cumulus cell-oocyte complexes in concentrations of dibutyryl cyclic adenosine monophosphate (dbcAMP) or 3-isobutyl-1-methyl xanthine (IBMX) (0.25 and 0.10 mM respectively) that suppressed spontaneous oocyte maturation without inducing cumulus expansion. These inhibitors also prevented the maximal induction of uncoupling that would have been provoked by biological grade preparations of either FSH or luteinizing hormone (LH). It was concluded that two factors were required to bring about maximal cumulus cell-oocyte uncoupling: one factor was dependent upon the action of gonadotropins on cumulus cell function, the other factor appeared to be a function of the oocytes, since maximal uncoupling could occur only after the germinal vesicles had broken down.     

毛囊分子调控机制

毛囊是产生毛被的组织,具有周期性,经历生长期、衰退期和休止期三个时期,毛囊与皮脂腺组成毛囊皮脂腺单位。本文从分子层面分别对调控毛囊周期性变化、毛囊皮脂腺发育以及毛囊中影响毛被光泽度及毡结度的因子等给予了详细阐述,这些均为提高山羊绒产量和质量提供了重要思路。     

Factors affecting the in vitro action of cumulus cells on the maturing mouse oocytes

The removal of cumulus cells (CCs) from oocytes at the germinal vesicle (GV) stage still represents a major limitation in such embryo techniques as GV transfer, somatic cell haploidization, and oocyte cryopreservation. However, no efficient in vitro maturation (IVM) system for CC-denuded oocytes (DOs) has been established in mammalian species. Although follicular cells are considered to play an important role in oocyte maturation, the specific role and mechanisms of action of different cell types are poorly understood. Reports on whether junctional association between CCs and the oocyte is essential for the beneficial effect of CC co-culture on oocyte maturation are in conflict. Our objective was to try to address these issues using the mouse oocyte model. The results indicated that while co-culture with the CC monolayer could only partially restore the developmental potential of DOs without corona cells, it restored the competence of corona-enclosed DOs completely. Culture in medium conditioned with CC monolayer also promoted maturation of DOs. However, co-culture with the monolayer of mural granulosa cells had no effect. The efficiency of CC co-culture was affected by various factors such as density and age of the CCs, the presence of gonadotropin in the maturation medium and the duration for in vivo (IVO) gonadotropin priming. It is concluded that mouse CCs produce a diffusible factor(s) that support DO maturation in a CC-oocyte junctional communication dependent manner. The data will contribute to our understanding the mechanisms by which CCs promote oocyte maturation and to the establishment of an efficient DO IVM system.     

Link protein as an enhancer of cumulus cell-oocyte complex expansion

To investigate the specific components involved in regulating cumulus cell-oocyte complex (COC) expansion in an in vitro mouse experiment, freshly-isolated COC were cultured in the presence of various combinations of FSH (1.0 microg/ml), proteins of the inter-alpha-inhibitor (I alpha I) family (a light chain, also known as bikunin, heavy chains [HC1 + HC2] and I alpha I [0.01-2.0 microg/ml]) and link protein (LP) (0.016-10 microg/ml) for 24 h and were observed for expansion of their cumulus cells (percent of COC with + 3 and + 4 expansion and average projected area). The COC were videotaped in real time at the initiation of culture and after 24 h of culture. FSH alone did not stimulate cumulus expansion under serum-free conditions; however, treatment with I alpha I (0.1-2.0 microg/ml) or heavy chains (10 microg/ml), but not bikunin (10 micro g/ml), in the presence of FSH significantly increased COC expansion, with maximal promotion occurring at 1.0 microg/ ml of I alpha I. Addition of LP (2.0 micro g/ml) to the medium containing I alpha I (1.0 microg/ml) and FSH resulted in significantly higher expansion levels than were observed in response to I alpha I alone, although LP alone (10 microg/ml) had no or very little effect by itself. Anti-I alpha I or anti-LP polyclonal antibody, which inhibits binding of I alpha I and LP, respectively, to hyaluronic acid (HA), markedly reduced expansion of the surrounding cumulus cell extracellular matrices. Therefore, in vitro, LP might serve, in part, to enhance the COC expansion possibly by stabilizing HA-I alpha I (or heavy chains) complex on the surrounding cumulus cell matrices.     

Developmental capability of denuded bovine oocyte in a co-culture system with intact cumulus-oocyte complexes: role of cumulus cells, cyclic adenosine 3',5'-monophosphate, and glutathione

Cumulus oophorus cells have been implicated in the regulation of female gamete development, meiotic maturation, and oocyte-sperm interaction. Nevertheless, the specific role of cumulus cells (CCs) during the final stages of oocyte maturation and fertilization processes still remains unclear. Several studies have been conducted in order to clarify the role of follicular cells using culture systems where denuded oocytes (DOs) were co-cultured with isolated CCs, or in the presence of conditioned medium. However, those attempts were ineffective and the initial oocyte competence to become a blastocyst after fertilization was only partially restored. Aim of the present study was to analyze the effect of the interactions between somatic cells and the female gamete on denuded oocyte developmental capability using a system of culture where CCs were present as dispersed CCs or as intact cumulus-oocyte complexes (COCs) in co-culture with oocytes freed of CC investment immediately after isolation from the ovary. Moreover, we analyzed the specific role of cyclic adenosine 3'-5' monophosphate (cAMP) and glutathione (GSH) during FSH-stimulated maturation of denuded oocyte co-cultured with intact COCs. Our data confirm that denuded oocyte has a scarce developmental capability, and the presence of dispersed CCs during in vitro maturation (IVM) does not improve their developmental competence. On the contrary, the co-presence of intact COCs during denuded oocyte IVM partially restores their developmental capability. The absence of CCs investment causes a drop of cAMP content in DOs at the beginning of IVM and the addition of a cAMP analog in the culture medium does not restore the initial oocyte developmental competence. The relative GSH content of denuded oocyte matured in presence of intact COCs is consistent with the partial recovery of their developmental capability. However, the complete restoration of a full embryonic developmental potential is achieved only when DOs are co-cultured with intact COCs during both IVM and in vitro fertilization (IVF). Our results suggest that the direct interaction between oocyte and CCs is not essential during IVM and IVF of denuded oocyte. We hypothesize that putative diffusible factor(s), produced by CCs and/or by the crosstalk between oocyte and CCs in the intact complex, could play a key role in the acquisition of developmental competence of the denuded female gamete.     

Production of cumulus expansion enabling factor by mouse oocytes grown in vitro: Preliminary characterization of the factor

The objective of this study was to determine whether fully grown oocytes, obtained after isolation from preantral follicles and growth in vitro, secrete paracrine factors affecting granulosa cell development and function. If so, the relative ease in producing oocytes in this way could facilitate the identification and characterization of the factors. As a test of this idea, the ability of in vitro grown oocytes to produce a paracrine factor that is known to enable the isolated cumulus oophorus to undergo expansion in response to follicle stimulating hormone (FSH) was determined. Initial experiments compared culture systems, which differed in the orientation of the oocyte-granulosa cell complexes from preantral follicles to an extracellular matrix, for their ability to support oocyte growth and the acquisition of competence to resume meiosis. The systems for culture on the surface of the matrix produced larger oocytes and the highest percentage of oocytes having competence to resume meiosis. Oocytes grown using this system secreted active cumulus expansion enabling factor, albeit at levels about half that of oocytes grown in vivo. A preliminary characterization of the cumulus expansion enabling factor secreted by the oocytes grown in vitro showed that activity was lost upon treatment with either heat (65°C for 15 min) or proteinase K. Activity did not pass through a membrane having a nominal molecular weight limit (NMWL) of 100 kd but did pass through a membrane having a NMWL of 300 kd. It is concluded that cumulus expansion enabling factor is secreted by oocytes grown in vitro. This factor is probably a protein or depends upon a protein for its activity. The ease in obtaining relatively large numbers of GVB-competent oocytes using techniques for growth in vitro combined with the demonstration that these produce cumulus expansion enabling factor indicates that these protocols can be used to produce oocytes for the collection and characterization of oocyte secretory products some of which are paracrine regulators of granulosa cells. © 1993 Wiley-Liss, Inc.     

Cumulus cells accelerate aging of mouse oocytes by secreting a soluble factor(s)

Control of oocyte aging in vitro is important for both human-assisted reproduction and animal embryo technologies because fertilization or artificial activation of aged oocytes results in abnormal development. Interactions between somatic and germ cells are also an important issue in current biological research. The role of cumulus cells (CCs) in maturation, ovulation, and fertilization of oocytes has been extensively studied, yet little is known about their role in oocyte aging. Although our previous study has shown that CCs accelerate the aging progression of mouse oocytes, the mechanism by which CCs accelerate oocyte aging is unknown. In this study, cumulus-denuded mouse oocytes (DOs) were co-cultured with cumulus-oocyte complexes (COCs) or CC monolayer or cultured in medium conditioned with these cells and changes in the susceptibility to activating stimuli and in MPF activity of oocytes were evaluated after different aging treatments. The results showed that culture with or in medium conditioned with COCs or CC monolayer promoted activation of DOs, indicating that a soluble factor is responsible for the aging-promoting effect. The in vivo and in vitro-matured DOs did not differ in responsiveness to the aging-promoting factor (APF). Heat shock did not accelerate oocyte aging unless in the presence of CCs. The production of APF was not affected by the age or maturation system of COCs, but increased with their density and duration of culture. The results strongly suggest that CCs accelerated oocyte aging by secreting a soluble APF into the medium. Further analysis showed that the APF was heat labile but stable to freezing, it had a threshold effective concentration and can be depleted by DOs.     

Configurations of germinal vesicle (GV) chromatin in the goat differ from those of other species

Configuration of germinal vesicle (GV) chromatin has been studied and found correlated with the developmental competence of oocytes in several mammalian species. A common feature in the configuration of GV chromatin in the species studied so far is that the diffuse chromatin (the so called "NSN" pattern) condenses into a perinucleolar ring (the so called "SN" configuration) with follicular growth. However, no study has been published on the configuration of GV chromatin in the goat. Nor is it known whether the perinucleolar ring of condensed chromatin (CC) in an oocyte represents a step toward final maturation or atresia. Changes in configurations of GV chromatin and RNA synthesis during goat oocyte growth, atresia and maturation in vivo and in vitro were investigated in this study. Based on both the size of nucleoli and the degree of chromatin condensation, the GV chromatin of goat oocytes was classified into GV1 characterized by large nucleoli and diffuse chromatin, GV2 with medium-sized nucleoli and condensed net-like (GV2n) or clumped (GV2c) chromatin, GV3 with small nucleoli and net-like (GV3n) or clumped (GV3c) chromatin, and GV4 with no nucleolus but clumped chromatin. The results showed that (i) the configurations of GV chromatin in the goat differ from those of other species in that the chromatin did not condense into a perinucleolar ring; (ii) most of the goat oocytes are synchronized at the GV3n configuration before GVBD; (iii) the GVn pattern might represent a healthy state, but the GVc an atretic state; (iv) in both goats and mice, the GC-specific (Chromomycin A3, CMA3) and the AT-specific (Hoechst 33342) fluorochromes followed the same pattern of distribution in GV chromatin; (v) the nucleolar size decreased significantly with oocyte growth and maturation in vivo and in vitro; and (vi) goat oocytes began GVBD at 8 hr and had completed it by 20 hr after onset of estrus. The peculiar configuration of GV chromatin of goat oocytes can be a useful model for studies of morphological and functional changes of different nuclear compartments during the cell cycle and cell differentiation, and the functional differentiation between GV3n and GV3c might be used for reference to the question whether the "SN" configuration in other species inclines toward ovulation or atresia.     

P75NTR在绒山羊毛囊生长周期中的表达

实验旨在研究绒山羊（Capra hircus）毛囊生长相关基因的表达规律,为绒山羊分子育种提供参考。本实验采用RTPCR、组织免疫荧光、Western blot等方法,研究神经营养素受体P75NTR在辽宁绒山羊皮肤组织中的表达和分布情况。结果表明：在毛囊生长周期的三个时期,均监测到P75NTR mRNA及蛋白的存在,P75NTR的荧光信号在退行期要强于其他两个时期,且在退行期毛囊外根鞘细胞中检测到P75NTR的高表达。以上结果表明,P75NTRmRNA及蛋白的表达与毛囊生长周期变化有一定的相关性,P75NTR受体在绒山羊的毛囊周期性变化中发挥着重要的功能。     

Activation of cumulus cell SMAD2/3 and epidermal growth factor receptor pathways are involved in porcine oocyte-cumulus cell expansion and steroidogenesis

Several lines of evidence suggest that in mice the activation of SMAD2/3 signaling by oocyte secreted factors, together with epidermal growth factor receptor (EGFR) activation, is essential to induce cumulus expansion. Here we show that inhibition of EGFR kinase in follicle stimulating hormone (FSH)-stimulated porcine oocyte-cumulus cell complex (OCCs) strongly decreases hyaluronan (HA) synthesis and its retention in the matrix, as well as progesterone synthesis. Although porcine cumulus cells undergo expansion independently of oocytes, we use biochemical and gene expression analyses to show that they do require activation of SMAD2/3 for optimal stimulation of HA synthesis and proteins involved in the organization of this polymer in the expanded matrix. Furthermore, FSH-induced progesterone synthesis by porcine cumulus cells was increased by blocking SMAD2/3 activation. In conclusion, these results support the hypothesis that an FSH-EGF autocrine loop is active in porcine OCCs, and provide the first evidence that the SMAD2/3 signaling pathway is induced by paracrine/autocrine factors in porcine cumulus cells and is involved in the control of both cumulus expansion and steroidogenesis.