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PubMed2006

Cyclooxygenase-2-derived prostaglandin E(2) directs oocyte maturation by differentially influencing multiple signaling pathways.

The process of oocyte maturation, which impacts ovulation and fertilization, is complex and requires an integration of the endocrine, paracrine, juxtacrine, and autocrine signaling pathways. This process involves an intimate interaction between the oocyte and encircling cumulus cells within a follicle, a unique venue for somatic and germ cell communication. Cumulus cell expansion and resumption of meiosis with germinal vesicle breakdown are major events in oocyte maturation. Cyclooxygenase-2 (COX-2)-derived prostaglandin E(2) (PGE(2)) is a known critical mediator of oocyte maturation, but the diverse function of this lipid mediator in oocyte maturation, ovulation, and fertilization has not been fully appreciated. We show here that gonadotropins in coordination with PGE(2) signaling via its cell surface G-protein-coupled EP2 and EP4 receptor subtypes direct cumulus cell expansion and survival and oocyte meiotic maturation by differentially impacting cAMP-dependent protein kinase, MAPK, NF-kappaB, and phosphatidylinositol 3-kinase/Akt pathways. This study is unique in the sense that it provides evidence for new site- and event-specific involvement of these signaling pathways under the influence of COX-2-derived PGE(2) during the critical stages of this somatic-germ cell interaction, an absolute requirement for oocyte maturation.

Animals↗
PubMed1997

Incidence of apoptotic bodies in membrana granulosa of the patients participating in an in vitro fertilization program.

OBJECTIVE: To investigate the incidence of apoptotic bodies in mural granulosa cell masses and cumulus cell masses. DESIGN: Nonrandomized, prospective study. SETTING: Department of Obstetrics and Gynecology, Yamagata University School of Medicine, Yamagata, Japan. PATIENT(S): One hundred twenty-nine normally ovulating women underwent ovulation induction for IVF-ET with GnRH analogue (GnRH-a) and gonadotropins. INTERVENTION(S): Patients underwent follicle aspiration after the administration of hCG. MAIN OUTCOME MEASURE(S): The nuclei of recovered granulosa cells were examined by fluorescence microscopy and the incidence of apoptotic bodies was tabulated. RESULT(S): The incidence of apoptotic bodies was significantly higher in mural granulosa cell masses than in cumulus cell masses in the entire group of 129 patients. Both incidence of apoptotic bodies of mural granulosa cell masses and cumulus cell masses were significantly higher in patients with less than six follicular oocytes compared with patients with six or more oocytes. Nonpregnant patients showed significantly higher incidence of apoptotic bodies in mural granulosa cell masses compared with pregnant patients. CONCLUSION(S): These results indicate that mural granulosa cell masses and cumulus cell masses may have different functions in follicular maturation. The incidence of apoptotic bodies in mural granulosa cell masses can be used as an indicator of success of IVF.

Adult↗
PubMed2000

Expression of pituitary adenylate cyclase activating polypeptide (PACAP) and PACAP type I A receptor mRNAs in granulosa cells of preovulatory follicles of the rat ovary.

Pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalamus and known to stimulate the production of cAMP in anterior pituitary cells. In the recent report, the expression of PACAP was detected in preovulatory follicles, and treatment with PACAP stimulated the production of progesterone and prostaglandin E(2) through the action of AC and PLC pathways in the ovary. PACAP binds to three type receptors. Type I A receptor is coupled to adenylate cyclase (AC) and phospholipase C (PLC) pathways, while type I B and type II receptors are only coupled to AC. Thus, the present study aimed to evaluate the temporal expression of PACAP and its type I A receptor mRNAs in the rat ovary after treatment with pregnant mare's serum gonadotropin and human chorionic gonadotropin (hCG). Northern blot analysis showed that PACAP transcripts were transiently expressed from 3-9 hr after hCG treatment, reaching a maximum at 6 hr. During these time points, PACAP mRNAs were specifically and strongly expressed in granulosa cells and cumulus cells of large preovulatory follicles and interstitial glandular cells. Type I A receptor mRNAs were also transiently expressed in granulosa cells of large preovulatory follicles from 3-9 hr after hCG treatment. PACAP and its type I A receptor mRNAs were expressed in the same preovulatory follicles. These results demonstrate that PACAP acts as an autoregulator or pararegulator through type I A receptor in granulosa cells and cumulus cells of large preovulatory follicles. Thus, we suggest that PACAP may have a critical role in granulosa cells of preovulatory follicles for the preparation of ovulation.

Animals↗
PubMed2006

Comparative studies on the mRNA expression of development-related genes in an individual mouse blastocyst with different developmental potential.

The evaluation of embryo morphology, widely used for selecting mammalian embryos before transfer, is not an adequate standard for selecting nuclear-transferred (NT) embryos. To search for markers useful for predicting the potential of NT embryos to develop into young, we examined the relation between the morphology of embryos with different developmental potential and gene expression of Oct 4, Nanog, Stat3, FGF4, Stella, and Sox2. In the present study, we examined pronuclear-exchanged blastocysts and morula blastomere, embryonic stem (ES) cell, and cumulus cell NT blastocysts, and in vivo-developed and in vitro-developed blastocysts. Based on the small variations in the gene expression levels among the in vivo-developed blastocysts, and the significant differences in gene expression between in vivo-developed (high developmental potential), and ES cell and cumulus cell NT blastocysts (low developmental potential), down-regulation of Sox2 and Oct4 genes is considered to be a candidate marker for the low potential of NT embryos to develop into young.

Animals↗
PubMed1998

Comparative analysis of calf and cow oocytes during in vitro maturation.

To determine possible causes of reported differences between developmental competence of oocytes isolated from prepubertal (10- to 14-week-old calves) and adult cows, three parameters were analysed, comparatively, during in vitro maturation (IVM): (1) oocyte diameter, (2) oocyte energy metabolism, and (3) protein synthesis of oocytes and cumulus cells. Cumulus-oocyte complexes were isolated from follicles of 3-5 mm in diameter in both age groups. Mean oocyte diameter was smaller (P < 0.02) in calves than in cows (118.04 +/- 1.15 versus 122.83 +/- 0.74 microns). During the first 3 hr of IVM, calf oocytes metabolised glutamine and pyruvate at lower rates than adult oocytes, but after 24 hr of culture, both molecules were metabolised at the same rate as for adult oocytes. A significant decrease in protein synthesis, as measured by [35S]methionine and [35S]cysteine incorporation was recorded after 9 hr of IVM in calf oocytes, while in adult oocytes a significant decrease in protein synthesis was detected only after 24 hr. After the first 3 hr of maturation, proteins of 130, 26, and 24 kDa were more abundant in adult than in calf oocytes, while a protein of 55 kDa was more visible in calf than in adult oocytes. At the same time, among proteins newly synthesised by cumulus cells, molecules of 405, 146, 101, and 77 kDa were more abundant in adults than in calves. In conclusion, calf oocytes and cumulus cells showed several differences when compared with their adult counterparts, which are consistent with their reported lower developmental competence.

Aging↗
PubMed1984

Selective binding of human cumulus cell-secreted glycoproteins to human spermatozoa during capacitation in vitro.

The results of this study demonstrate that glycoproteins manufactured by human cumulus cells can be detected bound to human spermatozoa incubated in capacitational medium containing the labeled cumulus-cell secretions. Cumulus-cell-secreted glycoproteins were labeled with a mixture of 3H-methionine and 3H-tryptophan or with 3H-fucose, and the binding of the labeled compounds to spermatozoa was evaluated by autoradiography. The binding was highly selective, involving only approximately 1% of the samples of spermatozoa used. The results suggest that the binding of cumulus-cell-secreted glycoproteins to spermatozoa may represent a final and highly selective step in human sperm capacitation.

Adult↗
PubMed1996

Human granulosa cells in culture exhibit functional cyclic AMP-regulated gap junctions.

Numerous gap junctions exist between granulosa cells, between cumulus cells and between cumulus cells and the oocyte. They may play a role in the regulation of both follicular development and oocyte status. We used primary cultures of human granulosa cells to study the molecular nature and functionality of these gap junctions. As shown by a cinemicrographic technique, during the first 3 days of culture, cells flattened and extended in several directions by means of cytoplasmic extensions. An ultrastructural study showed the presence of both intercellular and annular gap junctions after 48 h of culture. As revealed by immunodetection analyses, connexin 43 was present. An analysis using a functional procedure, the gap fluorescence recovery after photobleaching (FRAP) method, indicated that: (i) diffusional communication existed among granulosa cells; (ii) the communication was delayed by treatment with 1-heptanol, a well-documented inhibitor of gap junction permeability; and (iii) permeability was up-regulated by incubation with 8-Br-cAMP, an analogue of cyclic AMP. The detection of connexin 43 and functional gap junctions in networks of cytoplasmic extensions indicated junction formation among cells during culture. In conclusion, our results show that human granulosa cells in culture exhibited functional gap junctions. Connexin 43 was present and the permeability of the gap junctions was up-regulated by cyclic AMP, an important modulator of human granulosa cell function.

Alcohols↗
PubMed1988

The role of cumulus cell-secreted proteins in the development of human sperm fertilizing ability: implication in IVF.

Cumulus cells surrounding pre-ovulatory human oocytes were found to secrete a variety of proteins which became firmly associated with the cumulus intercellular material. Antibodies raised against human cumuli oophori completely blocked fertilization in vitro by impairing the sperm-zona pellucida interaction. A group of glycoproteins of high mol. wt were identified as the main cumulus cell secretory products. These proteins showed a marked affinity for human spermatozoa and were potent stimulators of the conversion of human and boar proacrosin into acrosin and of human sperm acrosome reaction. Another fraction of proteins of human cumulus intercellular matrix with an apparent mol. wt of approximately 25,000 daltons was also found to stimulate significantly the acrosome reaction of human spermatozoa, although this fraction had no proacrosin-converting activity. These results indicate that proteins secreted by pre-ovulatory human cumulus cells have an indispensable role in the development of human sperm fertilizing ability. This effect seems to be realized by a concerted action of different types of cumulus-derived proteins just prior to and during the sperm-zona pellucida interaction. Disorders of cumulus cell secretory activity may account for some cases of idiopathic infertility and repeated IVF failures.

Antibodies↗
PubMed1996

Proteoglycans and proteins in the extracellular matrix of mouse cumulus cell-oocyte complexes.

In the preovulatory follicle, the oocyte is surrounded by approximately 1000 closely associated cumulus cells forming the compact form of the cumulus cell-oocyte complex (COC). In response to the gonadotropin surge, the COC in a follicle destined for ovulation undergoes expansion when the cumulus cells synthesize and organize an extensive extracellular matrix enriched in hyaluronan. Successful expansion of the COC appears to be essential for ovulation and ultimately for fertilization. We studied this process in vitro by isolating compact COCs from preovulatory mouse follicles and incubating them under conditions which promote COC expansion by retention of newly synthesized hyaluronan (HA in the extracellular matrix around the cells. [3H]-Leucine and [35S]sulfate were used as precursors to label macromolecules synthesized by the cells that may be necessary for organizing the HA in this matrix. After labeling, expanded COCs were washed to remove medium and any labeled molecules that were not associated with the matrix. Macromolecules selectively associated with the matrix were then solubilized by digesting the expanded COCs briefly with Streptomyces hyaluronidase, an enzyme that specifically cleaves HA. Cells were removed by centrifugation, and the digest supernate was analyzed by molecular sieve chromatography and SDS-PAGE. A dermatan sulfate proteoglycan of large hydrodynamic size ( > 1 million Da) and a approximately 46-kDa protein were the predominant labeled species identified. The proteoglycan has properties similar to proteoglycans such as aggrecan and versican which interact specifically with HA. The approximately 46-kDa protein has the same molecular size as the link protein which interacts with HA and HA-binding proteoglycans to form stable ternary complexes in a variety of extracellular matrices. We propose that the dermatan sulfate proteoglycan and the approximately 46-kDa protein synthesized by the cumulus cells form similar ternary complexes that are necessary for retaining HA in the COC matrix and hence are required for successful COC expansion.

Animals↗
PubMed2006

Leptin promotes meiotic progression and developmental capacity of bovine oocytes via cumulus cell-independent and -dependent mechanisms.

Leptin has been shown to exert positive effects during the maturation of bovine oocytes, influencing blastocyst development, apoptosis, and the transcript levels of developmentally important genes. The present study was conducted to characterize further the mechanisms of leptin action on oocytes and the role of cumulus cells (CCs) in this context. In the first series of experiments, cumulus-oocyte complexes (COCs) were matured in serum-free medium that contained 0, 1 or 10 ng/ml leptin or in medium that was supplemented with 10% (v/v) estrus cow serum (ECS). Leptin concentrations of 1 and 10 ng/ml stimulated the meiotic progression of oocytes. Moreover, TUNEL staining demonstrated that these leptin doses reduced the proportion of apoptotic CCs. In the second series of experiments, COCs or denuded oocytes (DOs) were matured in the presence of 0 or 10 ng/ml leptin. The percentages of COCs and DOs with extruded polar bodies were increased by leptin. In contrast, positive effects of leptin on fertilization rates and blastocyst development were only observed after treatment of COCs but not of DOs. Leptin treatment of COCs consistently enhanced blastocyst development even after parthenogenetic activation of oocytes or after the removal of CCs before fertilization. The proportion of polyspermic oocytes was not affected by leptin treatment or oocyte denudation. In the third series of experiments, COCs were matured in the presence of 0, 1 or 10 ng/ml leptin. The transcript levels of specific genes were determined by reverse transcriptase-quantitative PCR (RT-qPCR) analysis of cumulus cells and single oocytes. Leptin treatment increased the levels of FAS, FASLG, and STAT3 transcripts in oocytes, but did not affect the LEPR, BAX, and BIRC4 mRNA concentrations. In cumulus cells, leptin treatment increased the mRNA levels for LEPR, STAT3, BAX, BIRC4, and FAS, but did not alter FASLG mRNA abundance. In conclusion, leptin differentially regulates gene expression in oocytes and cumulus cells. Moreover, leptin enhances both oocyte maturation and developmental capacity via cumulus cell-independent and -dependent mechanisms.

Animals↗
PubMed2000

Characterization of an immortalized human granulosa cell line (COV434).

We have investigated the biological characteristics of an immortalized granulosa cell line (COV434), which may be used to study follicular and oocyte maturation in vitro. Granulosa cell function was defined as consisting of three distinct properties: (i) production of 17beta-oestradiol in response to follicle stimulating hormone (FSH); (ii) presence of specific molecular markers of apoptosis enabling the induction of follicular atresia; and (iii) capacity to form intercellular connections with cells surrounding an oocyte. The addition of FSH to the culture medium supplemented with 10% fetal calf serum and 4-androstene-3,17-dione resulted in proliferation of the COV434 granulosa cells and in an increased synthesis of 17beta-oestradiol, indicating the presence of the FSH receptor and cytochrome P450 aromatase in these cells. The receptor for luteinizing hormone (LH) was undetectable. Similar expression of various apoptosis-associated genes was found in COV434 granulosa cells and in granulosa cells of patients stimulated with gonadotrophins for in-vitro fertilization, thus indicating that the immortalized COV434 granulosa cells were able to sustain apoptosis. Multiple intercellular connections were formed during co-culture of COV434 granulosa cells with cumulus cells containing an immature oocyte but not with cumulus cells devoid of an oocyte. Detailed morphological analysis of the intercellular connections with scanning electron microscopy and confocal light microscopy demonstrated the presence of long slender structures. It is concluded that the immortalized human granulosa cell line COV434 may be useful for experimental studies on follicular development.

Apoptosis↗
PubMed1981

Progesterone and prostaglandin secretion by ovulated rat cumulus cell-oocyte complexes.

Hormone production (progesterone and prostaglandin) by oocyte-cumulus cell complexes was studied in the postovulatory period. Cumulus-oocyte complexes were collected from oviducts of prepuberal rats after the synchronization of follicle development and ovulation with PMS gonadotropin and hCG injections. Cumulus masses from individual animals were cultured in roller vessels containing Eagles' Minimal Essential Media with or without added heat-inactivated rat sera (5% CO2-air; 37 C). Culture media were collected after 4.5 h of incubation and analyzed by RIA for prostaglandin E2 (PGE), PGF, and progesterone. Extensive progesterone secretion occurred during the culture period and was markedly stimulated by the addition of rat serum; however, cumulus masses did secrete some progesterone in the absence of rat serum and without exogenous gonadotropins. The addition of aminoglutethimide, a steroidogenesis inhibitor, suppressed progesterone secretion but did not alter the secretion of PGF or PGE. Both PGF and PGE were detected in the culture medium, and in all cases, PGE was the predominant PG detected. Indomethacin, an inhibitor of PG synthesis, suppressed PGE and PGF secretion but had no effect on progesterone synthesis. Small amounts of PGs were also detected in nonincubated cumulus-oocyte masses. Hormone secretion in culture was measured after cumulus mass dissociation by enzymatic digestion. Somatic cumulus cells, rather than germ cells, were the primary cellular source of progesterone and PGs. The results demonstrate that cumulus-oocyte complexes can synthesize and secrete steroid and PG hormones subsequent to ovulation, and thus, these masses should be considered functional endocrine tissues. The physiological significance of the endocrine activities of the cumulus-oocyte complex are discussed in terms of oviductal and gametic processes. (Endocrinology 108: 457, 1981)

Aminoglutethimide↗
PubMed1989

[Morphological analysis of resumption of meiosis observed in oocytes of mouse vesicular follicles].

Resumption of meiosis observed in vesicular follicles of immature 32-34 day-old mice was classified morphologically, and the morphological mechanism to induce resumption of meiosis was examined. Resumption of meiosis was observed in 22.1% of vesicular follicles, and the dominant stage of the meiotic division was the first or second metaphase. Cumulus-enclosed and cumulus-free oocytes were separated from ovaries by dissection of ovaries. All of the cumulus-enclosed oocytes contained germinal vesicle, however 57.9% of cumulus-free oocytes did not, suggesting that the disappearance of cumulus cells around oocytes precedes the induction of resumption of meiosis. About 80-90% of the oocytes with germinal vesicle formed first polar body after 15 hrs of incubation. After incubation of oocytes showing resumption of meiosis, but without the first polar body, 36% of the oocytes extruded the first polar body indicating that resumption of meiosis is associated with progression to the second metaphase. Morphological characteristics of the surrounding structures of oocytes showing resumption of meiosis was classified into three categories; extensive proliferation of granulosa cells, degeneration of cumulus cells and their disappearance around oocytes, and detachment of cumulus cell-oocyte complexes from granulosa cell layer. It was suggested that cell-to-cell interaction among cumulus cells, oocytes and granulosa cells was impaired at the time of resumption of meiosis. Granulosa cells secrete an inhibitor of the resumption of meiosis. The resumption of meiosis after the surge of luteinizing hormone follows cumulus dispersion which make oocytes independent from the influence of granulosa cells by the disruption of communication.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

[Studies on in vitro capacitation of goat spermatozoa by heparin treatment].

Systematical studies are lacking on the influencing factors and mechanisms of the heparin enhanced sperm capacitation, although many studies have shown that heparin enhanced sperm capacitation. The effect of heparin concentration and exposure time, incubation temperature and co-culture with oviductal epithelial cells or cumulus cells on goat sperm capacitation were investigated in this study. The motility, membrane and acrosome integrity and capacitated percentage of goat spermatozoa were assessed after different heparin treatments, and rates of fertilization and embryo cleavage were compared after in vitro insemination of oocytes with spermatozoa capacitated by different heparin treatments. The major results are summarized as follows: 1) When spermatozoa were capacitated with heparin at 5, 10, 25, 50 and 100 microg/mL for 45 min, 50 and 100 microg/mL heparin treatments produced the highest capacitated percentages of 55% and 56%, respectively, but the percentage of spermatozoa with intact acrosomes in the 100 microg/mL heparin treatment decreased significantly (P < 0.05) in comparison with that in the control group, indicating that the optimal heparin concentration for goat sperm capacitation would be 50 microg/mL. 2) Capacitated percentage of spermatozoa increased with extension of treatment time when goat sperm were treated with 50 microg/mL heparin for 0, 10, 20, 30, 45, 60 or 120 min. Although heparin treatments for 45 to 120 min did not differ significantly (P > 0.05) in capacitated sperm percentages, sperm motility and membrane integrity decreased significantly when treated with heparin for 120 min. This suggested that the optimal exposure time of heparin at 50 microg/mL for goat sperm capacitation would be 45 to 60 min. 3) Significantly higher capacitated percentages of spermatozoa were obtained when goat sperm were treated at 42 and 38.5 degrees C than at 15 and 37 degrees C, but sperm motility and acrosome integrity were significantly lower when spermatozoa were treated at 42 degrees C than they were treated at other temperatures. Temperature of 38.5 degrees C would, therefore, be the optimal temperature for goat sperm capacitation. 4) The capacitated percentage of spermatozoa was significantly higher when goat sperm were co-cultured with oviductal epithelial cells than when treated with heparin alone or co-cultured with cumulus cells, but sperm motility and membrane and acrosome integrity did not differ significantly among the three treatments. Rates of fertilization (91.3%) and cleavage (72.2%) were significantly higher in the oviductal epithelial cell co-culture group than those in the heparin alone group. This indicated that co-culture with oviductal epithelial cells significantly enhanced goat sperm capacitation by heparin treatment.

Acrosome Reaction↗
PubMed1995

Immunolocalization of transforming growth factor-beta 1 and transforming growth factor-beta 2 in the mouse ovary during gonadotrophin-induced follicular maturation.

An immunohistochemical approach was utilized to evaluate the cellular distribution of transforming growth factor-beta 1 (TGF beta 1) and transforming growth factor beta 2 (TGF beta 2) at different stages of follicle development in the prepubertal mouse ovary under the following conditions: (i) after pregnant mare's serum gonadotrophin (PMSG) treatment; (ii) after PMSG and human chorionic gonadotrophin (HCG) treatment; (iii) after PMSG and HCG treatment plus mating. In the immature ovary, TGF beta 1 and TGF beta 2 immunoreactivities are localized in theca and granulosa cells and in oocytes. After PMSG treatment, TGF beta 1 and TGF beta 2 immunoreactivities are localized in granulosa cells; in addition, TGF beta 2 staining is noted in the matrix surrounding antral cells. Staining for both TGR beta 1 and TGF beta 2 drops in the theca but persists in the oocyte. PMSG plus HCG treatment results in a significant increase in TGF beta 1 and TGF beta 2 immunoreactivity in the theca and in the maintenance of TGF beta 1 staining in both basal granulosa cells and cumulus cells whereas TGF beta 2 immunoreactivity is essentially localized in the matrix surrounding cumulus cells. Staining for TGF beta 1 and TGF beta 2 persists in the oocyte. Following PMSG plus HCG treatment and mating, TGF beta 1 immunoreactivity is localized in the luteal cells of corpora lutea and TGF beta 2 shows a similar localization pattern. This study provides evidence that TGF beta 1 and TGF beta 2 peptides are expressed in specific cell types during induced follicular maturation in the mouse ovary.

Animals↗
PubMed2005

Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer.

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic errors including abnormal DNA hypermethylation. Recently, we elucidated by using round spermatids that, after nuclear transfer, treatment of zygotes with trichostatin A (TSA), an inhibitor of histone deacetylase, can remarkably reduce abnormal DNA hypermethylation depending on the origins of transferred nuclei and their genomic regions [S. Kishigami, N. Van Thuan, T. Hikichi, H. Ohta, S. Wakayama. E. Mizutani, T. Wakayama, Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids, Dev. Biol. (2005) in press]. Here, we found that 5-50 nM TSA-treatment for 10 h following oocyte activation resulted in more efficient in vitro development of somatic cloned embryos to the blastocyst stage from 2- to 5-fold depending on the donor cells including tail tip cells, spleen cells, neural stem cells, and cumulus cells. This TSA-treatment also led to more than 5-fold increase in success rate of mouse cloning from cumulus cells without obvious abnormality but failed to improve ES cloning success. Further, we succeeded in establishment of nuclear transfer-embryonic stem (NT-ES) cells from TSA-treated cloned blastocyst at a rate three times higher than those from untreated cloned blastocysts. Thus, our data indicate that TSA-treatment after SCNT in mice can dramatically improve the practical application of current cloning techniques.

Animals↗
PubMed1988

Disruption of cellular associations within the granulosal compartment of periovulatory ovine follicles: relationship to maturation of the oocyte and regulation by prostaglandins.

Temporal relationships were defined in sheep between the onset of the preovulatory surge of luteinizing hormone (LH; 0 h), dispersion of mural granulosal cells and cumulus oophorus, resumption of meiosis of the oocyte, and ovulation. A quantitative increase in intercellular spacing among mural granulosal and cumulus cells was detected at the light-microscopic level 12 h following the onset of the surge of LH. Evidence of breakdown of the germinal vesicle of oocytes was apparent at 16 h and thereafter. Ovulation occurred near 24 h. Systemic administration of indomethacin, an inhibitor of prostaglandin/PG biosynthesis, at 8 h subsequently prevented dissociation of mural granulosal and cumulus cells and suppressed maturation of the oocyte. The action of the drug was reversed by intrafollicular injection of PGE2 at 12 h; PGF2 alpha was ineffective in this regard. Prostaglandin E2 appears to be involved in preovulatory ovine follicles in the dissolution of cell-to-cell contacts and in maturation of the oocyte.

Animals↗
PubMed2003

Expression of nitric oxide synthase-3 in porcine oocytes obtained at different follicular development.

The present study was designed to determine the localization of nitric oxide synthase-3 (NOS-3) in porcine follicles during follicular development. A 130-kDa NOS-3 protein was found with greater frequency much in the oocytes than in the cumulus cells, as revealed by Western blotting analysis. The content of NOS-3 in the oocyte was higher in large follicles (> 7-mm diameter) than in small follicles (< 2-mm). The data by Western blotting showed the same pattern as the observations obtained from the immunohistochemical studies, in which the periphery of the oocyte stained strong positive. The inner surface cell layer of granulosa cells and cumulus cells were positive staining, especially in large antral follicles. In the primordial follicles, NOS-3 was restricted to the cytoplasm of oocytes, and no stained product was observed in the nucleus of oocytes or granulosa cells. A significant synthesis of NO by oocytes was observed in the presence of ionomycin, but not in the absence of ionomycin, indicating that oocyte NOS-3 functions in response to transient elevations in the intracellular calcium level. We concluded that NOS-3 is expressed in the oocyte from the primordial follicular stage to antral follicular stage, and that it is functional at least in the antral follicles.

Animals↗