Effect of FSH and HCG on progesterone secretion by cultured oocyte-cumulus complexes and granulosa cells from porcine antral follicles: Influence of follicular development

Oocyte-cumulus complexes and granulosa cells were harvested from small (1–2 mm), medium (3–5 mm), and large (6–12 mm) porcine antral follicles and cultured for 2 and 3 days. The effects of various doses of purified hCG and human FSH on progesterone secretion and monolayer formation were examined. After a 2-day culture period it was found that FSH was more effective in stimulation of progesterone secretion by cultured oocyte-cumulus complexes than in granulosa cells harvested from small follicles (P < 0.01), whereas hCG was more effective in stimulating progesterone secretion in granulosa cells than in oocytecumulus complexes harvested from large follicles. In contrast, after a 3-day culture period, granulosa cells secreted more progesterone compared to oocytecumulus complexes under control conditions or in the presence of hCG or FSH. After 3 days both FSH and hCG stimulated progesterone secretion by oocytecumulus complexes and granulosa cells; however, the hormone effect was greater upon granulosa cells than oocyte-cumulus complexes. After 3 days of culture in the case of both follicular cell types, there was a greater response to FSH in the case of cells harvested from small compared to large follicles. The reverse was true in the case of hCG responsiveness. Monolayer formation ability of oocyte-cumulus complexes was greater in the case of complexes harvested from small and medium than complexes harvested from large follicles. Addition of hCG to the cultures led to a dose-dependent decrease in monolayer formation by oocyte-cumulus complexes harvested from all sizes of follicles.     

Gossypol inhibits follicle-stimulating hormone- and epidermal growth factor-stimulated expansion of oocyte-cumulus complexes from porcine preovulatory follicles

The role of gossypol in the cumulus expansion of oocyte-cumulus complexes (OCC) isolated from large antral porcine follicles was investigated. Marked suppression of cumulus expansion stimulated with follicle-stimulating hormone (FSH) and epidermal growth factor (EGF) was observed in the presence of different concentrations of gossypol. Comparable inhibitory effects were obtained in the presence of NO donor, S-nitroso-N-acetylpenicillamine or sodium nitroprusside, suggesting that the inhibitory effect of gossypol may be mediated via NO generation. The inhibitory effect of gossypol on cumulus expansion of OCC was accompanied by inhibition of progesterone secretion of OCC and the decrease of [125I]EGF binding to granulosa cells.     

Effect of gonadotropins and prostaglandin on cumulus mucification in cultures of intact follicles

Follicles of 28 day-old pregnant mare serum gonadotropin (PMSG)-primed rats, were cultured for up to 24 hours in the presence or absence of ovine gonadotropins, highly purified rat gonadotropins, dibutyryl cyclic AMP (dbcAMP), methylisobutylxanthine (MIX), choleratoxin (CT), or prostaglandin E2 (PGE2). The morphology of the cumulus-oocyte complexes isolated from these follicles was subsequently examined with the light microscope. Cumulus mucification was studied under the different culture conditions using scanning electron microscopy (SEM) and the hyaluronidase sensitivity test. The features of the cumulus-oocyte complexes in the control cultures did not change throughout the incubation period, while complexes from follicles incubated with LH, FSH, dbcAMP, MIX, CT, or PGE2 changed their appearance and accumulated extracellular mucoid material. Treatment of these cumuli with hyaluronidase resulted in lysis of the extracellular mucus and dispersal of the cumulus masses. The results of this study agree with our earlier observation that the maturation of the cumulus-oophorus, which occurs in vivo following the LH surge, can be induced in vitro by either gonadotropins or cAMP. Prostaglandin E2 did not affect cumulus cells, unless incubated enclosed by their follicles. This suggests that this hormone may influence the cumulus cells indirectly, probably via other components.     

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.     

Components of cigarette smoke inhibit expansion of oocyte-cumulus complexes from porcine follicles

The role of alkaloids in cigarette smoke was investigated in the cumulus expansion of oocyte-cumulus complexes (OCC) isolated from large antral porcine follicles. Suppression of the cumulus expansion stimulated by FSH was observed in the presence of different concentration of cadmium, anabasine and nicotine but not its metabolite cotinine. There were comparable inhibitory effects of cadmium and nicotine on the synthesis and accumulation of hyaluronic acid in the cell/matrix compartment of OCC. The inhibitory effect of tested compounds on the cumulus expansion was accompanied by decreased progesterone synthesis by cumulus cells during 42 h incubation of OCC with FSH. The results suggest that cigarette smoking may affect intrafollicular processes, which are responsible for normal ovulation and fertilization.     

Epidermal growth factor-receptor tyrosine kinase activity regulates expansion of porcine oocyte-cumulus cell complexes in vitro

We have recently shown that epidermal growth factor (EGF) strongly stimulates expansion of porcine oocyte-cumulus complexes (OCCs) isolated from large follicles (>6 mm) and does not promote expansion of OCCs from small (3-4-mm) follicles. In order to elucidate the role of EGF in OCCs expansion, in the present study, we first examined the presence of EGF receptors (EGFRs) in cumulus cells isolated from follicles of different sizes. Surprisingly, immunoblotting showed that cumulus cells obtained from all follicular size categories contained similar amounts of EGFR protein. On the other hand, we found a dramatic difference in the pattern of protein tyrosine phosphorylation in a comparison of cumulus cells isolated from small and large follicles treated by EGF. Furthermore, tyrosine-phosphorylated EGFR was specifically immunoprecipitated with antiphosphotyrosine antibodies from EGF-treated cumulus cells isolated from the large follicles. This result strongly indicates that only OCCs from the large follicles contain mature EGFRs that are capable of becoming activated by EGF. Remarkably, preincubation of cumulus cells from small follicles (3-4 mm) with FSH strongly increased EGF-stimulated tyrosine phosphorylation to levels comparable with OCCs from large follicles. The FSH-dependent activation of EGFRs was beneficial for expansion of OCCs isolated from the small follicles since OCCs treated sequentially by FSH (3 h) and EGF (1 h) underwent expansion significantly better then OCCs cultured in FSH or EGF alone. We conclude that a FSH-dependent pathway has an important role in the maturation of the EGFR in cumulus cells and that activation of EGFR-dependent signaling is sufficient to induce expansion.     

Covalent transfer of heavy chains of inter-alpha-trypsin inhibitor family proteins to hyaluronan in in vivo and in vitro expanded porcine oocyte-cumulus complexes

Previous studies have shown that the heavy chains (HCs) of serum-derived inter-alpha-trypsin inhibitor (IalphaI) molecules become covalently linked to hyaluronan (HA) during in vivo mouse cumulus expansion and significantly contribute to cumulus matrix organization. Experiments with mice suggest that the incorporation of such proteins in cumulus matrix appears to be rather complex, involving LH/hCG-induced changes in blood-follicle barrier and functional cooperation between cumulus cells, granulosa cells, and oocyte within the follicle. We demonstrate here that HC-HA covalent complexes are formed during in vivo porcine cumulus expansion as well. Western blot analysis with IalphaI antibody revealed that follicular fluids from medium-sized follicles and those from large follicles unstimulated with hCG contain high levels of all forms of IalphaI family members present in pig serum. The same amount of HCs were covalently transferred from IalphaI molecules to HA when pig oocyte-cumulus complexes (OCCs) were stimulated in vitro with FSH in the presence of pig serum or follicular fluid from unstimulated or hCG-stimulated follicles. In addition, HC-HA coupling activity was stimulated in cumulus cells by FSH treatment also in the absence of oocyte. Collectively, these results indicate that IalphaI molecules can freely cross the blood follicle barrier and that follicular fluid collected at any stage of folliculogenesis can be successfully used instead of serum for improving OCC maturation. Finally, pig cumulus cells show an autonomous ability to promote the incorporation of IalphaI HCs in the cumulus matrix.     

Mouse oocytes promote proliferation of granulosa cells from preantral and antral follicles in vitro.

Evidence is now emerging that the oocyte plays a role in the development and function of granulosa cells. This study focuses on the role of the oocyte in the proliferation of (1) undifferentiated granulosa cells from preantral follicles and (2) more differentiated mural granulosa cells and cumulus granulosa cells from antral follicles. Preantral follicles were isolated from 12-day-old mice, and mural granulosa cells and oocyte-cumulus complexes were obtained from gonadotropin-primed 22-day-old mice. Cell proliferation was quantified by autoradiographic determination of the 3H-thymidine labeling index. To determine the role of the oocyte in granulosa cell proliferation, oocyte-cumulus cell complexes and preantral follicles were oocytectomized (OOX), oocytectomy being a microsurgical procedure that removes the oocyte while retaining the three-dimensional structure of the complex or follicle. Mural granulosa cells as well as intact and OOX complexes and follicles were cultured with or without FSH in unconditioned medium or oocyte-conditioned medium (1 oocyte/microliter of medium). Preantral follicles were cultured for 4 days, after which 3H-thymidine was added to each group for a further 24 h. Mural granulosa cells were cultured as monolayers for an equilibration period of 24 h and then treated for a 48-h period, with 3H-thymidine added for the last 24 h. Oocyte-cumulus cell complexes were incubated for 4 h and then 3H-thymidine was added to each group for an additional 3-h period. FSH and/or oocyte-conditioned medium caused an increase in the labeling index of mural granulosa cells in monolayer culture; however, no differences were found among treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of luteinizing hormone and progesterone on maturation and fertilizability of rat oocytes in vitro

The effects of luteinizing hormone (NIH-bovine LH) and progesterone on maturation in vitro of oocyte-cumulus complexes from adult proestrous rats were studied by comparing proportions of oocytes showing germinal vesicle breakdown, mucification of the cumulus oophorus, and fertilizability. Addition of either or both of the hormones to the medium in concentrations between 1.25 and 10 μg/ml during maturation had no discernible effect on germinal vesicle breakdown or on fertilization. Mucification was stimulated by LH and even more by LH plus progesterone. It was concluded that maturation in vivo is the result of concerted action of the two hormones. However, addition of LH + progesterone had no effect on the fertilizability of these oocytes. We attribute this to a relative insensitivity of the system for fertilization in vitro to subtle changes in the oocyte.     

Follicular fluid modulation of functional LH receptor induction in pig granulosa cells

Follicular fluid was collected from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) follicles of pigs, treated with charcoal to remove steroids, and tested for effects on the induction of functional LH/hCG receptors in cultures of granulosa cells from small antral pig follicles. Granulosa cells were cultured for 2, 4 or 6 days in Medium 199 + 10% pig serum. Granulosa cells cultured in the presence of purified human FSH (0.1 microgram/ml, LER 8/117), insulin (1 mU/ml), cortisol (0.01 microgram/ml) and thyroxine (10(-7) M) accumulated a 4- to 8-fold increase in LH/hCG receptors compared to control cultures. The amounts of cyclic AMP and progesterone secreted after exposure to ovine LH (1 microgram/ml: NIH-S19) were also increased 2-3-fold and 80-100-fold, respectively. Exposure to FSH alone resulted in lower amounts of LH/hCG receptors with a concomitant decrease in optimum LH responses. Addition of 12.5-50% follicular fluid obtained from small (1-2 mm) follicles led to a dose-dependent inhibition of the FSH plus insulin, cortisol and thyroxine induction of LH/hCG receptors after 4 days of culture. Fluid from medium follicles showed reduced ability to inhibit LH/hCG receptor induction, and fluid from large follicles exerted only a slight inhibition or no inhibition of receptor induction. Fluid from medium-sized and large follicles exerted a progressive dose-dependent stimulation of progesterone secretion by the granulosa cell cultures. The inhibitory activity was precipitated primarily with 70% ethanol and to a lesser degree by 36 and 90% ethanol. These studies demonstrate that induction of functional LH/hCG receptors in cultures of pig granulosa cells from immature follicles is enhanced by including insulin, cortisol and thyroxine, in addition to FSH, in the culture medium, and that follicular fluid modulates both receptor induction and progesterone secretion as a function of follicular maturation.     

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.     

Meiotic state of bovine oocytes is regulated by interactions between cAMP,cumulus, and granulosa

Bovine oocytes are arrested at the prophase of first meiotic cell cycle. Meiosis resumes in oocytes of pre-ovulatory follicles upon LH surge. However, oocytes from secondary follicles spontaneously resume meiosis in the absence of hormones if removed from the follicle and cultured in vitro. The nature of meiotic arrestor in bovine follicles is poorly understood. In this study we investigated the role of cell-cell interactions between granulosa and cumulus cells and the oocyte in mediating maintenance of meiotic arrest by cAMP. We sorted oocytes as granulosa-cumulus oocyte complexes (GCOC) if surrounded with cumulus cells attached to a large granulosa investment or cumulus oocytes complexes (COC) if surrounded with cumulus cells only and investigated the role cAMP in maintenance of meiotic arrest in these oocytes under various conditions. In hormone- and serum-free medium both GCOC and COC enclosed oocytes resumed meiosis. When [cAMP](i) was elevated with addition of invasive adenylate cyclase (iAC) GCOC enclosed oocytes were maintained in the prophase with intact germinal vesicle (GV) while COC enclosed oocytes underwent GV breakdown (GVBD). iAC elevated [cAMP](i) in both types of oocytes to the same level. If oocytes were liberated from the cumulus and granulosa cells, they re-initiated meiosis in serum and hormone free medium, but remained in the GV stage if iAC was added to the medium. Untreated GCOC and COC enclosed oocytes extruded first polar body at the same frequency in hormone-supplemented media. GCOC and COC enclosed oocytes but not denuded oocytes (DO) cultured without somatic cells acquired developmental competence if cultured in hormone-containing medium. It is concluded that maintenance of meiotic arrest is regulated by the interplay of [cAMP](i), and cumulus and granulosa cells.     

Stimulatory action of follicular fluid components on maturation of granulosa cells from small porcine follicles

Follicular fluid obtained from large (6-12 mm) porcine follicles (LFF) was investigated to determine its stimulatory activity on progesterone secretion and on follicle stimulating hormone (FSH) induction of 125I-human chorionic gonadotropin (hCG)-luteinizing hormone (LH) binding sites in porcine granulosa cells in a 4-day culture. Incubation of granulosa cells harvested from small porcine follicles (1-2 mm) with 50% LFF led to stimulation of LH/hCG binding sites and progesterone secretion. After partial purification of pooled LFF or proteins precipitated with 90% ethanol on Sephadex G-100 the greatest stimulatory activity was found in the second protein peak eluted from the column. Chromatography of part of the active fraction on DEAE Sephacel using a continuous gradient of NH4HCO3 yielded seven protein fractions. The second fraction, which eluted early, contained the majority of the stimulatory activity which was purified about 32-fold compared to native LFF. In contrast, addition of follicular fluid recovered from small porcine follicles inhibited FSH induction of LH/hCG binding sites and progesterone secretion. It can be concluded, that maturation of granulosa cells from small follicles may be enhanced by protein(s) present in LFF, but not in fluid recovered from less mature follicles.     

Steroidogenesis by the human oocyte-cumulus cell complex in vitro

The ability of the human oocyte-cumulus cell complex to synthesize progesterone, androgens and estrogens and to modify its endocrine environment in vitro was investigated. Germinal-vesicle stage oocytes with adhering layers of cumulus cells were recovered from human ovaries and maintained for 40–50 h in vitro in a culture medium with or without antral fluid.The results show that oocyte-cumulus (0-C) cell complexes were capable of synthesizing progesterone, androgens and estrogens. Oocytes with the capacity of resuming meiosis in vitro were part of an 0-C complex producing significantly more progesterone than those 0-C complexes containing oocytes incapable of resuming meiosis. Irrespective of the stage of oocyte maturation at the end of culture, testosterone and estrone were respectively the major androgen and estrogen produced.It is concluded that the oocyte-cumulus compartment of the antral follicle is a steroidogenically competent unit and that it has the capacity to modify the endocrine microenvironment of the follicle.     

Oocyte-dependent activation of mitogen-activated protein kinase (ERK1/2) in cumulus cells is required for the maturation of the mouse oocyte-cumulus cell complex

Luteinizing hormone (LH) induces maturational processes in oocyte-cumulus cell complexes (OCC) of preovulatory follicles that include both resumption of meiosis in the oocyte and expansion (mucification) of the cumulus oophorus. Both processes require activation of mitogen-activated protein kinase (MAPK) in granulosa cells. Here, it is reported that inhibition of MAPK activation prevented gonadotropin-stimulated resumption of meiosis as well as the rise in expression of two genes whose products are necessary for normal cumulus expansion, Has2 and Ptgs2. However, inhibition of MAPK did not block gonadotropin-induced elevation of granulosa cell cAMP, indicating that the activation of MAPK required for inducing GVB and cumulus expansion is downstream of cAMP. Moreover, activation of MAPK in cumulus cells requires one or more paracrine factors from the oocyte to induce GVB and cumulus expansion; MAPK activation alone is not sufficient to initiate these maturational processes. This study demonstrates a remarkable interaction between the oocyte and cumulus cells that is essential for gonadotropin-induced maturational processes in OCC. By enabling gonadotropin-dependent MAPK activation in granulosa cells, oocytes promote the generation of a return signal from these cells that induces the resumption of meiosis. It also appears that an oocyte-dependent pathway downstream from oocyte-enabled activation of MAPK, and distinct from that promoting the resumption of meiosis, governs cumulus expansion.     

Intrafollicular control of oocyte maturation in the PIG

Summary The mammalian oocyte becomes arrested at the diplotene stage of the first meiotic division during prenatal or early postnatal life. It remains arrested in meiosis until shortly before ovulation when the surge of gonadotropin induces resumption and completion of meiosis to the metaphase II stage. When oocytes are harvested from medium-sized or large follicles of pig and other species and cultured, they resume meiosis spontaneously indicating that the follicles exert an inhibitory influence on meiosis. To analyze the control of meiosis by follicular components, culture of isolated pig oocytes in the presence of follicular cells or follicular fluid (FF1) has been used as a model in this laboratory. An oocyte maturation inhibitor (OMI) has been isolated and partially purified by ultrafiltration and gel chromatography of FF1 and shown to be a polypetide with a molecular weight in the order of 2000 daltons. Physiological characterization has shown that the effect of OMI in vitro is reversible and that it can be overcome by luteinizing hormone (LH). The action of OMI requires the presence of cumulus cells surrounding the oocyte since it was found that denuded oocytes, stripped of cumulus cells, do not respond to OMI. Furthermore, when cumulus-enclosed oocytes were cultured, OMI inhibited the differentiation of the cumulus cells in terms of morphology and progesterone secretion in a dose-related manner. The inhibition of cumulus differentiation by OMI was reversible and could be overcome by LH. The results indicate that the effect of partially purified OMI upon meiosis may be mediated by the cumulus cells. Presented in the formal symposium on Sexual Differentiation in Vitro and in Vivo at the 29th Annual Meeting of the Tissue Culture Association, Denver, Colorado, June 4–8, 1978. This study was supported by Grants 760–0530 from the Ford Foundation (to C.P.C.), and Grant B78-14F-5158-01 from the Swedish Medical Research Council (to T.H.).     

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.     

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.     

Follicle population, cumulus mucification, and oocyte chromatin configuration during the periovulatory period in the female dog

This study was designed to describe the follicular population present on the canine ovary (Canis familiaris) during the preovulatory period and essentially the changes in oocyte size, mucification, and chromatin configuration occurring from before the luteinizing hormone (LH) surge up to postovulation. In a first experiment, ovaries of beagle bitches were collected before (n = 21) or after LH surge but before ovulation (post-LH surge/preovulation stage, n = 24) as determined using hormone (LH, estradiol, progesterone) assays and ultrasonography. All large (>2 mm) follicles were measured and punctured. The numbers of oocytes collected per follicle and the degree of cumulus mucification were recorded. In a second experiment, ovaries were similarly collected before (n = 13) and after the LH surge but before ovulation (n = 11) as well as after ovulation as determined by ultrasonography (n = 9). Chromatin configuration of the oocytes was observed by DNA staining and confocal microscopy. In Experiment 1, before the LH peak, an average of 13.5 ± 0.7 follicles per bitch (total 284 follicles) were detected, and the maximal follicle diameter reached 6.5 mm. Large follicles were observed already in this period of the cycle and as early as when progesterone was still below 0.5 ng/mL. After the LH peak but before ovulation, 11.0 ± 0.7 follicles were present (total 264 follicles). Fully mucified cumulus cells were observed only in follicles larger than 4 mm. Multi-oocytic follicles represented 7% (before LH peak) and 4% (after LH peak) of the follicular population. In Experiment 2, all the oocytes were at the germinal vesicle (GV) stage, but three chromatin configurations could be distinguished: diffuse, partly grouped, and fully grouped chromatin. The proportion of oocytes with fully grouped chromatin increased with the follicular diameter and the time in estrus, the maximum being observed after the LH peak. These results suggest that (1) before LH peak, follicles are already of large diameter, similar to the ones at ovulation; (2) the ability for cumulus mucification is acquired during the late steps of follicular growth; (3) three GV patterns may be observed during the periovulatory period.     

Administration of GnRH in vivo stimulates progesterone and inhibits androgen accumulation by ovarian follicles isolated from pubertal rabbits

A single i.v. injection of gonadotropin releasing hormone (GnRH) to pubertal female rabbits led to an ovulatory pulse of LH but no ovulations resulted. By contrast, 5 i.v. injections over 6 h led to 1-3 ovulations in 5 of 8 animals treated. Twenty-four hours after the initial injection animals were killed and follicles isolated. Large follicles greater than 1 mm dia, from both GnRH treated groups released more progesterone during the control incubation period than those from saline treated. Small follicles less than 1 mm dia, from the same GnRH groups accumulated 3-6 times more progesterone than those from saline treated when stimulated with luteinizing hormone (LH). Testosterone accumulation by small and large follicles was not affected by one injection of GnRH but was depressed in follicles from rabbits treated with 5 injections of GnRH. A single injection of GnRH enhanced the ability of small and large follicles to release estradiol which was depressed 30% in the presence of LH. Multiple GnRH injections depressed estradiol accumulation by small and large follicles. These data suggest the administration of GnRH in vivo can have stimulatory as well as inhibitory effects on subsequent follicular steroid release and accumulation in vitro.