Presence Of Granulosa Cells Research Articles

Effect of cumulus cells and duration of culture on nuclear maturation of oocytes of pubertal and pre-pubertal individuals from Sus scrofa domesticus

Objective: to evaluate the nuclear maturation ability of pig oocytes in relation to the morphology of the surrounding cumulus cells, the duration of in vitro maturation (IVM) and the physiological status of cumulus-oocyte complexes (COCs) donor animals.Materials and methods. СOCs collected from cycling (pubertal) and non-cycling (prepubertal) pigs ovaries were categorized according to the number of cumulus cell (CC) layers: COC I – more then 4, COC II – 3-4, COC III – 1-2 and СOC with granulosa cells (GCOC). Oocytes within COCs were matured during the first 22 h of culture in the TC-199 medium with 25 mM HEPES, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.4% BSA, 10% follicular fluid, 0.5 μg/mL FSH, 0.5 μg/mL LH and 50 μg/mL gentamicin sulfate and thereafter in medium of the same composition but without hormones. After 42, 44, 46, 48 hours of culture the maturation of oocytes of all categories and age groups was assessed by the sign of their release of the first polar body. Results. It was shown that at least 42 hours of in vitro culture of pubertal pig oocytes are required to ensure a high level of nuclear maturation. The duration of the in vitro maturation was found to be 44-48 hours for pre-pubertal pigs. Oocytes surrounded by 1-2 layers of CC, when cultured for less than 48 hours, are inferior in nuclear maturation to oocytes with a more multilayered cumulus. The presence of GC (granulosa cells) around COC retards retards the progression of nuclear maturation when cultured for less than 42 hours in prepubertal and 44 hours in pubertal gilts. Oocytes from cycling pigs reach peak maturation earlier than those from non-cycling gilts and can be used in assisted reproductive technologies (ART). Conclusions. It was concluded that both morphology of the surrounding cumulus cells and physiologic status of COC donor animals should be considered when choosing the duration of pig oocytes culture for the purpose of their in vitro maturation.

PSX-A-24 Late-Breaking: Testosterone production by theca cells of large preovulatory follicles is affected by growth hormone depending on the hen age and the presence of granulosa cells

Abstract Testosterone produced by theca cells may be involved in regulating of the growth and ovulation of hen preovulatory follicles (Rangel, Gutierrez, Gen Comp Endocrinol, 203:250, 2014). In the current research, we studied effects of growth hormone (GH), a known regulator of the hen ovarian function, on in vitro testosterone production by the theca layer (TL) from the two largest yellow follicles in relation to the hen age and the presence of the granulosa layer (GL). Young hens with long clutch (YLC, 32–33 week-old, >10 eggs per clutch) and old hens with short clutch (OSC, 74–76 week-old, 3–6 eggs per clutch) were used. After isolation, TL from F1 and F2 follicles (n = 8–9) was cultured for 18 h in two systems, separately or together with the corresponding GL, in the presence or absence of chicken GH (25 ng/ml). Concentrations of testosterone in the spent media were measured by ELISA. The data were analyzed by RM-ANOVA. In the case of separate TL culture, GH did not change significantly testosterone production in both follicles of YLC hens and reduced it from 338±105 to 152±52 fmol/mg tissue (P < 0.05) in F1 follicles of OSC hens. When TL was cultured in the presence of GL, GH enhanced 1.8-2.6-fold (P < 0.05) the secretion of testosterone in the case of F1 follicles and decreased it 1.8-2.5-fold (P < 0.05) in the case of F2 follicles in both young and old hens. Regardless of the treatment, follicular size or culture system, the production of testosterone in OSC hens was 2–5 times higher than in YLC hens. The results indicate that the interaction between TL and GL changes the steroidogenic response of theca cells from preovulatory follicles to GH in young and old hens. Furthermore, testosterone production is obviously increased with reproductive aging of laying hens. The study was supported by RFBR (19-016-00216).

Encircling granulosa cells protects against di-(2-ethylhexyl)phthalate-induced apoptosis in rat oocytes cultured in vitro.

The present study investigated if the presence of encircling granulosa cells protected against di(2-ethylhexyl)phthalate (DEHP)-induced oxidative stress in rat oocytes cultured in vitro. Denuded oocytes and cumulus-oocyte complexes (COCs) were treated with or without various doses of DEHP (0.0, 25.0, 50.0, 100, 200, 400 and 800 μM) in vitro. Morphological apoptotic changes, levels of oxidative stress and reactive oxygen species (ROS), mitochondrial membrane potential, and expression levels of apoptotic markers (Bcl2, Bax, cytochrome c) were analyzed. Our results showed that DEHP induced morphological apoptotic changes in a dose-dependent manner in denuded oocytes cultured in vitro. The effective dose of DEHP (400 µg) significantly (P>0.05) increased oxidative stress by elevating ROS levels and the mitochondrial membrane potential with higher mRNA expression and protein levels of apoptotic markers (Bax, cytochrome c). Encircling granulosa cells protected oocytes from DEHP-induced morphological changes, increased oxidative stress and ROS levels, as well as increased expression of apoptotic markers. Taken together our data suggested that encircling granulosa cells protected oocytes against DEHP-induced apoptosis and that the presence of granulosa cells could act positively towards the survival of oocytes under in vitro culture conditions and may be helpful during assisted reproductive technique programmes.

Hormonal and cellular interactions in follicular steroid biosynthesis by the sheep ovary

Studies of isolated cell types from sheep follicles revealed several functional changes which occur during follicular maturation. Cyclic AMP production by granulosa cells from the smallest follicles studied (1-3 mm diameter) was stimulated by FSH but not by hCG, suggesting functional FSH receptors at this early stage of differentiation. Medium-sized follicles (4-6 mm) responded to both FSH and hCG. Granulosa cells were unable to synthesize androgens, but readily converted exogenous testosterone to oestradiol-17 beta. This conversion occurred to a limited extent in the cells from the smallest follicles, but was much greater in medium and large (greater than 6 mm) follicles. Oestradiol production by theca preparations from small follicles was barely detectable, but increased markedly with increasing follicle size. Androgen (androstenedione and testosterone) production by theca preparations was stimulated by hCG. This stimulation was short-lived, and levels declined to below control values after 6 h of culture. This decline could not be prevented by addition of cyclic AMP. The presence of granulosa cells with thecal preparations (i.e. follicle wall tissue) enhanced production of androgen by the theca, the effect being more marked for testosterone than for androstenedione. In-vivo studies in which granulosa cells and follicular fluid were removed during the preovulatory period suggested that granulosa cells and/or follicular fluid contributed to the oestradiol secreted into the ovarian vein during this period, but did not exclude a significant contribution by the theca as well.

172 Expression of proliferation and apoptosis markers in cumulus cells surrounding matured and aged oocytes exposed to luteotropic factors during the second phase of in vitro maturation

The quality and developmental capacity of mammalian oocytes depends on cooperation with surrounding cumulus cells. The functional state and activity of cumulus cells changes with oocyte maturation, especially during the oocyte transition from metaphase I (MI) to metaphase II (MII) stage. In the present work, effects of 3 luteotropic factors, progesterone (P4), prolactin (PRL), and LH, during the second phase of in vitro maturation (IVM) on the subsequent expression of proliferation and apoptosis markers in bovine cumulus cells surrounding matured and aged oocytes were studied. A total of 1532 cumulus-oocyte complexes (COC) were cultured for 12h in TCM-199 containing 10% fetal calf serum (FCS), 10μg mL−1 porcine FSH, and 10μg mL−1 ovine LH at 38.5°C and 5% CO2. Thereafter, COC were transferred to the following IVM systems: (1) TCM-199 containing 10% FCS (Control 1) and (2) a monolayer of granulosa cells (GC) precultured for 12h in TCM-199 containing 10% FCS (Control 2). In both systems, the medium of experimental groups was supplemented with either P4 (50 ng mL−1) or bovine PRL (50ng mL−1) or ovine LH (5μg mL−1); then, the COC were matured for next 12h. Half of the COC matured for 12h in both systems were cultured for an additional 24h in fresh TCM-199 containing 10% FCS to test long-term hormonal effects during oocyte aging. After culture, the cumulus expression of the proliferation marker proliferating cell nuclear antigen (PCNA) and the pro-apoptotic markers caspase-3 and Bax was assessed by the immunocytochemical method. The data from 4 to 5 replicates using 84 to 106 COC per treatment were analysed by ANOVA. After IVM in System 1, the rate of PCNA-positive cumulus cells was higher (P<0.05) in the PRL-treated group (41.3±1.6%) than in the control (34.6±2.3%) or LH-treated group (29.9±2.9%), but did not differ from that in the P4-treated group (38.2±4.8%). In the presence of GC (System 2), the respective rates did not change but were more variable. Aging of COC matured in both systems led to a 1.4- to 1.9-fold reduction in the proportion of the cells containing the proliferation marker PCNA (P<0.05). Meanwhile, none of the hormones tested had any long-term effect on the proliferative activity of senescent cumulus cells. The rate of cumulus cells expressing caspase-3 in different groups varied from 48.5±4.9 to 53.8±5.8% and did not depend on the hormones, IVM system, or oocyte aging. The proportion of the Bax-positive cells was also unaffected by luteotropic factors but increased 1.4 to 1.6 times (P<0.01) following 24h of COC aging. Our findings indicate that PRL can exert a short-term stimulatory action on the proliferative activity of bovine cumulus cells in the course of the second phase of IVM. Meanwhile, the cumulus expression of pro-apoptotic markers caspase-3 and Bax is not responsive to P4, PRL, or LH during the second step of IVM. The study was supported by the Russian Science Foundation (project 16-16-10069).

132 The Developmental Competence of Bovine Oocytes Exposed to Progesterone and Luteotrophic Hormones During the Second Step of Two-Step Maturation

Data on effects of progesterone (P4) during in vitro maturation of bovine oocytes on their capacity for embryonic development are contradictory. Our study was aimed at characterising effects of P4 and 2 luteotropic hormones, prolactin (PRL) and LH, on bovine oocyte developmental competence during the second step of two-step maturation (from metaphase (M)I to MII). Slaughterhouse-derived cumulus-enclosed oocytes (CEO) were matured for 12 or 24 h [one-step (OS) Control] in TCM-199 containing 10% fetal calf serum (FCS), 10 μg mL−1 porcine FSH, and 10 μg mL−1 ovine LH at 38.5°C and 5% CO2. The CEO cultured for 12 h were transferred to the following culture systems: (1) TCM-199 containing 10% FCS (Control 1) or (2) a monolayer of granulosa cells (GC) precultured for 12 h in TCM-199 containing 10% FCS (Control 2); then, the oocytes were matured for next 12 h. In both systems, the medium of experimental groups was supplemented with either P4 (50 ng mL−1) or bovine PRL (25 and 50 ng mL−1) or ovine LH (5 μg mL−1). All treatments were repeated 5 to 6 times using 138 to 196 oocytes per group. Following IVM, all oocytes underwent IVF as described previously (Singina et al. 2014 Reprod. Fertil. Dev. 26, 154). Embryos were cultured in CR1aa medium until Day 5 post-insemination and then transferred to the same medium supplemented with 5% FCS and cultured to Day 7. Embryo development was evaluated at Days 2 and 7 for cleavage and blastocyst formation. Apoptosis was detected by the TUNEL method using 26 to 47 blastocysts per group (from 4 to 5 separate experiments). For each system, arcsine-transformed data were analysed by one-way ANOVA. In OS Control, the cleavage and blastocyst rates were 68.9 ± 4.4% and 22.0 ± 2.4%, respectively. Regardless of the system or medium of two-step culture, the cleavage rate did not differ from that for OS Control, varying between 57.6 and 68.4%. In the absence of GC (System 1), the blastocyst yield in the P4 group (30.4 ± 0.8%) was greater (P < 0.05) than in OS Control and Control 1 (20.2 ± 2.7%) as well as in the groups treated with LH (19.1 ± 3.0%) and 25 ng mL−1 PRL (20.1 ± 2.7%). In the presence of GC, P4 raised the yield from 16.7 ± 2.3% (Control 2) to 27.7 ± 2.4% (P < 0.05). Furthermore, in System 2, the blastocyst rate in groups treated with P4 and 50 ng mL−1 PRL (25.0 ± 2.8%) was higher (P < 0.05) than in the LH group (13.9 ± 2.6%). Meanwhile, the proportion of apoptotic nuclei (2.3-6.9%) was not associated with the system of oocyte maturation or effects of hormones studied. Our data indicate that P4 (50 ng mL−1) can enhance the developmental competence of bovine oocytes during the second step of two-step maturation regardless of the presence of granulosa cells, whereas the similar effect of PRL (50 ng mL−1) is less pronounced and depends on the granulosa-conditioned environment. This research was supported by the Russian Science Foundation (project 16-16-10069).

Effects of retinoic acid on maturation of immature mouse oocytes in the presence and absence of a granulosa cell co-culture system

Evaluation of the all-trans retinoic acid (t-RA) effects on in vitro maturation (IVM) and in vitro fertilization (IVF) of immature mouse oocytes in the presence and absence of granulosa cell monolayer. Denuded oocytes isolated from mice ovaries and matured in IVM medium alone (Control I), IVM medium in the presence of granulosa cells (Control II), IVM medium with t-RA (Experimental I) and IVM medium simultaneously with t-RA and granulosa cells (Experimental II). After 24h, matured oocytes were fertilized in T6 medium and their development was followed until the blastocyst stage. Metaphase II oocytes ploidy were evaluated by chromosome counting. The t-RA group compared to the control groups showed no obvious abnormalities. Additionally maturation and embryo development rates significantly increased in the t-RA treated granulosa cell co-culture system. In conclusion, association of t-RA with granulosa cell co-culture during in vitro maturation increases meiosis resumption, formation of metaphase II oocytes, as well as 2-cell and blastocyst stage embryos.

Role of granulosa and theca cell interactions in ovarian follicular maturation

We developed a culture system in which two types of ovarian follicular cells were allowed to attach to opposite sides of a collagen membrane. Using this in vitro cell culture system, we studied the effects of granulosa- and theca-cell interaction on the morphology, structure, and function of bovine ovarian follicular cells. In the first part of the study, we explored how the interaction between theca and granulosa cells affects the morphology and structure of the cells. This study was done using follicular cells collected from bovine ovarian follicles at the early developmental stage. Granulosa cells cultured alone were flattened, and formed a monolayer sheet. By contrast, granulosa cells cultured with theca cells were convex, and formed multilayer sheets. Theca cells cultured alone were thin, flat, and spindle-shaped. Theca cells cultured with granulosa cells were also spindle-shaped; however, they appeared convex and more densely packed when compared with theca cells cultured alone. In the second part of the study, the possible role of the cellular interaction in the control of differentiation and growth of granulosa and theca cells was investigated. When follicular cells were isolated from the early stage of follicular development, theca cells reduced progesterone and inhibin production by granulosa cells and augmented the growth of granulosa cells. When the cells were isolated from the late stage of follicular development, by contrast, theca cells augmented hormonal production by granulosa cells, and did not affect the growth of granulosa cells. The growth and androstenedione production by theca cells were increased by the presence of granulosa cells, irrespective of the origin of follicular cells. These results demonstrated that communication between two types of follicular cells results in reciprocal modulation of their morphology, structure, growth, and function. Cellular interactions seem to be one of the major factors controlling the differentiation and growth of the follicular cells during the follicular maturation process. In contrast to granulosa and theca cells cultured alone, cells in the coculture seemed to possess morphological and functional characteristics more similar to those of cells in the growing follicular wall in vivo. Thus, we speculate that the interaction between these two types of follicular cells is essential for the maintenance of original structure and function of the bovine follicular wall.

Theca cells and theca-cell conditioned medium inhibit the progression of FSH-induced meiosis of bovine oocytes surrounded by cumulus cells connected to membrana granulosa.

The effect of follicular cells and their conditioned media on the FSH-induced oocyte maturation of oocytes surrounded by cumulus cells connected to the membrana granulosa (COCGs) was investigated. COCGs and cumulus oocyte complexes (COCs) were cultured for 22 hr in M199 supplemented with 0.05 IU FSH/ml in either the presence of pieces of theca cell layer or in the presence of pieces of membrana granulosa. COCGs and COCs were also cultured for 22 hr in either theca-cell conditioned medium (CMt) or in granulosa cell conditioned medium (CMg), both supplemented with 0.05 IU FSH/ml. To investigate the importance of cell-cell contacts between granulosa cells and cumulus cells, oocytes were cultured as COCs in CMt, as COCs in CMt supplemented with pieces of membrana granulosa, or as COCGs in CMt. In all groups the medium was supplemented with 0.05 IU FSH/ml. After culture the nuclear status of the oocytes was assessed using orcein staining. Culture of COCGs in the presence of theca cells as well as in CMt resulted in a significantly decreased proportion of oocytes that had undergone germinal vesicle breakdown (GVBD) at the end of the culture period as compared to the control. Of the oocytes that resumed meiosis in the presence of theca cells or in CMt, the proportion of oocytes that progressed up to the MII stage was significantly reduced. This indicates the production of a meiosis-inhibiting factor by theca cells. Culture with COCs instead of COCGs resulted in comparable results although the effect was less pronounced. The significant effect on the progression of meiosis of oocytes cultured as COCGs or as COCs, obtained in the presence of granulosa cells or in CMg, was much weaker than the effect of theca cells or culture in CMt. Culture of COCs in CMt supplemented with layers of membrana granulosa and 0.05 IU FSH/ml, resulted in significantly less oocytes that resumed meiosis as compared to culture of COCs in CMt. Of the oocytes that showed GVBD, the proportion that progressed up to the MII stage was significantly reduced. Attachment of the COCs to the membrana granulosa enhanced this inhibiting action of CMt on the progression of meiosis. It is concluded that theca cells secrete a stable factor that inhibits the progression of FSH-mediated meiosis in oocytes of COCGs.

Effect of insulin‐like growth factor I and its interaction with gonadotropins on in vitro maturation and embryonic development, cell proliferation, and biosynthetic activity of cumulus‐oocyte complexes and granulosa cells in buffalo

In this study we have examined the effect of insulin like growth factor I (IGF-I) and its interaction with gonadotropins in the presence or absence of granulosa cell coculture on in vitro oocyte maturation (IVM) and their subsequent embryonic development in buffalo. We also have examined the role of IGF-I alone or in combination with gonadotropins on DNA synthesis, steroidogenesis, and protein synthesis of cumulus-oocytes complexes (COCs) and granulosa cells. Results showed that IGF-I stimulates oocytes maturation in a dose-dependent manner, with maximal effect at a dose of 100 ng/ml (P < 0.05). IGF-I showed positive interaction with follicle-stimulating hormone (FSH) in the presence or absence of granulosa cells on meiotic maturation and synergistically enhanced DNA synthesis, protein synthesis, and steroidogenesis in the presence of granulosa cells. This synergistic effect is mainly caused by the increase of IGF-I receptors in granulosa cells by FSH, as evident by [125I]IGF-I binding study. Luteinizing hormone (LH), however, was found to suppress IGF-I and IGF-I + FSH stimulated oocyte maturation. Addition of LH to cultures containing IGF-I + FSH, on the contrary, caused a significant increase in oocyte maturation when cocultured with granulosa cells. Addition of IGF-I during IVM significantly improve cleavage and blastocyst development rate over the control group. However, there was no cumulative effect when IGF-I and gonadotropins were present together. Addition of granulosa cells during IVM, however, enhanced blastocyst development in the IGF-I + FSH and IGF-I + FSH + LH groups. Our results demonstrated that IGF-I is a major follicular factor responsible for stimulating oocyte maturation in the buffalo. Interaction between IGF-I and FSH suggests that they seem to act synergistically as an autocrine and paracrine regulator of granulosa cells and therefore together promote mitosis, steroidogenesis, and protein synthesis. Mol. Reprod. Dev. 49:277–285, 1998. © 1998 Wiley-Liss, Inc.

Effect of insulin-like growth factor I and its interaction with gonadotropins on in vitro maturation and embryonic development, cell proliferation, and biosynthetic activity of cumulus-oocyte complexes and granulosa cells in buffalo.

In this study we have examined the effect of insulin like growth factor I (IGF-I) and its interaction with gonadotropins in the presence or absence of granulosa cell coculture on in vitro oocyte maturation (IVM) and their subsequent embryonic development in buffalo. We also have examined the role of IGF-I alone or in combination with gonadotropins on DNA synthesis, steroidogenesis, and protein synthesis of cumulus-oocytes complexes (COCs) and granulosa cells. Results showed that IGF-I stimulates oocytes maturation in a dose-dependent manner, with maximal effect at a dose of 100 ng/ml (P < 0.05). IGF-I showed positive interaction with follicle-stimulating hormone (FSH) in the presence or absence of granulosa cells on meiotic maturation and synergistically enhanced DNA synthesis, protein synthesis, and steroidogenesis in the presence of granulosa cells. This synergistic effect is mainly caused by the increase of IGF-I receptors in granulosa cells by FSH, as evident by [125I]IGF-I binding study. Luteinizing hormone (LH), however, was found to suppress IGF-I and IGF-I + FSH stimulated oocyte maturation. Addition of LH to cultures containing IGF-I + FSH, on the contrary, caused a significant increase in oocyte maturation when cocultured with granulosa cells. Addition of IGF-I during IVM significantly improve cleavage and blastocyst development rate over the control group. However, there was no cumulative effect when IGF-I and gonadotropins were present together. Addition of granulosa cells during IVM, however, enhanced blastocyst development in the IGF-I + FSH and IGF-I + FSH + LH groups. Our results demonstrated that IGF-I is a major follicular factor responsible for stimulating oocyte maturation in the buffalo. Interaction between IGF-I and FSH suggests that they seem to act synergistically as an autocrine and paracrine regulator of granulosa cells and therefore together promote mitosis, steroidogenesis, and protein synthesis.

Effect of β-mercaptoethanol supplementation during in vitro maturation of pig oocytes on blastocyst development after in vitro fertilization

The effects of granulosa cells in maturation media on male pronuclear formation and in vitro development of in vitro-matured and fertilized (IVM-IVF) bovine oocytes were examined. In Experiment 1, cumulus-oocyte complexes (COCs) were aspirated from follicles of slaughterhouse ovaries and classified into 4 morphological categories according to the surrounding cumulus cells: Grade 1 (> 4 layers), Grade 2 (3 to 4 layers), Grade 3 (1 to 2 layers) and Grade 4 (denuded). Oocytes were co-cultured with or without granulosa cells (1 × 106 cells/ml) for 21 to 22 h. At 18 and 192 h after insemination, the abilities of oocytes to form a male pronucleus and develop up to the blastocyst stage in vitro were determined, respectively. The presence of granulosa cells during maturation did not affect (P < 0.05) the ability of oocytes in Grades 1 and 2 to form a male pronucleus and to develop to the blastocyst stage in Grades 1 and 4. However, the incidence of male pronuclear formation in Grades 3 and 4 and in vitro development to the blastocyst stage in Grades 2 and 3 was higher (P < 0.05) when COCs were cultured in the presence of granulosa cells than when cultured in the absence of granulosa cells. In Experiment 2, COCs collected by ultrasound-guided aspiration were co-cultured with or without granulosa cells, fertilized and cultured as described above. The incidence of blastocysts at 192 h after insemination was higher (P < 0.05) when COCs were cultured for maturation in the presence of granulosa cells (24%) than in the absence of granulosa cells (12%). These results demonstrate that supplementation of maturation medium with granulosa cells improves the quality of oocytes with relatively few cumulus cell layers, as determined by male pronuclear formation and in vitro development. We also conclude that this supplementation effectively improves the developmental ability of bovine IVM-IVF oocytes that were collected by ultrasound-guided transvaginal aspiration.

Influence of granulosa cells and of different somatic cell types on mammalian oocyte development in vitro.

In mammals the ability of an oocyte to become fertilised is the result of a complex process occurring within the ovarian follicle which depends on the stagespecific expression of oocyte genes and the presence of granulosa cells (for a review see Buccioneet al., 1990a). The coordinated development of germinal and somatic components of the follicle is regulated by two principal systems of interaction, based on the presence of gap junctions and on the production of paracrine factors. Gap junctions link granulosa cells to each other and to the oocyte (Anderson &amp;amp; Albertini, 1976), and represent a major route for the transfer of small molecules involved in oocyte metabolism (for a review see Mangiaet al., 1992) and regulation of the arrest and resumption of meiosis (for a review see Eppig, 1993). The production of paracrine factors by granulosa cells has been suggested by the findings that these cells express the production of theSteellocus, the Steel factor (SLF) or kit ligand (KL; Motroet al., 1991; Manovaet al., 1993), and that this factor promotes oocyte growthin vitrowhen used at high concentrations (Packeret al., 1994). Since KL is too large to be transmitted through gap junctions, it must necessarily be released in the extracellular environment before binding to the c-kitreceptor present on oocyte membrane (Manovaet al., 1990; Horieet al., 1991).

Covalent Linkage between Proteins of the Inter-α-inhibitor Family and Hyaluronic Acid Is Mediated by a Factor Produced by Granulosa Cells

The direct interaction of hyaluronic acid (HA) and proteins of the inter-alpha-inhibitor family plays a critical role in organization and stabilization of the expanding cumulus extracellular matrix (cECM) following an ovulatory stimulus. Despite similarities in the morphology of cumulus oocyte complexes (COCs) expanding in vivo and in vitro, we find that the cECM of COCs which expand within intact follicles are more elastic and resistant to shear stress than the cECM of those stabilized in vitro. Western blot analysis shows that only the heavy chains of inter-alpha-inhibitor are incorporated into the cECM and appears to be covalently linked to HA after stabilization in vivo while intact inter-alpha-inhibitor is bound to the HA-enriched cECM by a non-covalent mechanism in in vitro stabilized COCs. However, purified pre-alpha-inhibitor and HA can form covalent linkage in the presence of granulosa cells or with granulosa cell-conditioned medium. In addition, COCs resistance to shear stress is also enhanced by coincubation with granulosa cells. Upon formation of the apparent covalent linkage between heavy chains and HA in culture medium, the light chain (bikunin) is concomitantly released into the medium as a complex with chondroitin sulfate moieties of inter-alpha-inhibitor supporting the possibility that HA may replace the chondroitin sulfate linkage to the heavy chains. We speculate that a factor(s) secreted by granulosa cells within the follicle may catalyze a transesterification reaction resulting in an exchange of chondroitin sulfate with HA at the heavy chain/chondroitin sulfate junction followed by release of chondroitin sulfate-bikunin into the follicular fluid. It is also possible that the consequent further stabilization of the cECM through the covalent interaction of HA and heavy chains of inter-alpha-inhibitor may play an important role in the process of ovulation.

Presence of granulosa cells during oocyte maturation improved in vitro development of IVM-IVF bovine oocytes that were collected by ultrasound-guided transvaginal aspiration

The effects of granulosa cells in maturation media on male pronuclear formation and in vitro development of in vitro-matured and fertilized (IVM-IVF) bovine oocytes were examined. In Experiment 1, cumulus-oocyte complexes (COCs) were aspirated from follicles of slaughterhouse ovaries and classified into 4 morphological categories according to the surrounding cumulus cells: Grade 1 (> 4 layers), Grade 2 (3 to 4 layers), Grade 3 (1 to 2 layers) and Grade 4 (denuded). Oocytes were co-cultured with or without granulosa cells (1 × 10 6 cells/ml) for 21 to 22 h. At 18 and 192 h after insemination, the abilities of oocytes to form a male pronucleus and develop up to the blastocyst stage in vitro were determined, respectively. The presence of granulosa cells during maturation did not affect (P < 0.05) the ability of oocytes in Grades 1 and 2 to form a male pronucleus and to develop to the blastocyst stage in Grades 1 and 4. However, the incidence of male pronuclear formation in Grades 3 and 4 and in vitro development to the blastocyst stage in Grades 2 and 3 was higher (P < 0.05) when COCs were cultured in the presence of granulosa cells than when cultured in the absence of granulosa cells. In Experiment 2, COCs collected by ultrasound-guided aspiration were co-cultured with or without granulosa cells, fertilized and cultured as described above. The incidence of blastocysts at 192 h after insemination was higher (P < 0.05) when COCs were cultured for maturation in the presence of granulosa cells (24%) than in the absence of granulosa cells (12%). These results demonstrate that supplementation of maturation medium with granulosa cells improves the quality of oocytes with relatively few cumulus cell layers, as determined by male pronuclear formation and in vitro development. We also conclude that this supplementation effectively improves the developmental ability of bovine IVM-IVF oocytes that were collected by ultrasound-guided transvaginal aspiration.

Presence of granulosa cells during oocyte maturation improved in vitro development of IVM-IVF bovine oocytes collected by ultrasound-guided transvaginal aspiration

Frozen-thawed ejaculated stallion spermatozoa were preincubated for 3 h in BO medium containing 5 mM caffeine and then treated with 0.1 μ M calcium ionophore A23187 for 60 sec. Aliquots of the sperm suspension (final concentration 1–2 × 107/ml) were added to the oocytes which had been matured in vitro for 32 h. In Experiment 1, there were 3 groups of oocytes; cumulus intact, denuded zona-intact, and zona-free. Cumulus cells were removed with 0.5% hyaluronidase and the zona pellucida with 0.1% protease. The oocytes were fixed 20 h after insemination with acetic acid:ethanol (1:3) and stained with 1% orcein. The sperm penetration rate of zona-free oocytes was 83%, whereas the sperm penetration rate was very low (1 to 3%) in the cumulus-enclosed or zona-intact oocytes. In Experiment 2, denuded zona-intact oocytes were placed in PBS supplemented with 10% fetal bovine serum 1 h before the end of in vitro maturation. The zona pellucida was micromanipulated with a metal microblade under × 100 magnification within 20 min of treatment with 0.3 M sucrose. For partial zona dissection, a slit in the zona pellucida was made. For partial zona removal, oocytes were transferred to protein-free PBS to fix the oocytes on the bottom of the Petri-dish and to remove a piece of the zona pellucida. Micromanipulated oocytes were subjected to in vitro fertilization as described above. Zona-intact and zona-free oocytes treated with sucrose solution for 20 min were used as controls. The penetration rates were 4 (257), 12 (758), 52 (3160), and 86% (4451) for zona-intact, partially zona dissected, partially zona removed, and zona-free oocytes, respectively. Proportions of oocytes with monospermic penetration were 100 (22), 57 (47), 58 (1831), and 34% (1544), respectively. In Experiment 3, sperm penetration and male pronucleus formation in the partially zona removed oocytes were examined at 2.5 to 20.0 h of insemination. Sperm penetration started 2.5 h post-insemination (22%, 1149), and increased to 38% (2155) at 5 h, to 46% (2350) at 10 h, and to 56% (2748) at 20 h. The transformation of sperm heads into male pronuclei was first observed 10 h post insemination. These results indicate that assisted fertilization techniques may be a useful tool for achieving fertilization and embryo production in vitro in horses.

In vitro maturation of oocytes from a marsupial, the tammar wallaby (Macropus eugenii)

This study examined the competence of oocytes from the tammar wallaby, Macropus eugenii to mature in vitro. Oocytes were collected from follicles > 1 mm diameter 24 h after pregnant mare serum gonadotrophin (PMSG) treatment and incubated in Eagle's minimum essential medium supplemented with 10% fetal calf serum, at 35 degrees C in 5% CO2 in air for 24, 36, or 48 h. Oocytes were incubated either granulosa cell-intact or granulosa cell-free or in the presence of 10 IU ml-1 PMSG or 10 micrograms ml-1 porcine luteinizing hormone (LH) + 10 micrograms ml-1 porcine follicle stimulating hormone (FSH). The ability of oocytes recovered from small (< 1.5-mm-diameter) and large (> or = 1.5 mm-diameter) follicles to mature in vitro was also examined. The nuclear status of oocytes was assessed using the DNA-specific dye Hoechst 33342. Initially, all oocytes examined contained a germinal vesicle. After 24 h of culture, 60% of oocytes had progressed to metaphase I or anaphase I. After 36 h, approximately 20% of oocytes possessed metaphase II chromosomes, and 20% of oocytes were at metaphase I or anaphase I. At the completion of the 48 h culture period, 40% of oocytes had completed maturation to the metaphase II stage. In vitro oocyte maturation after 48 h was not affected by the presence of granulosa cells, PMSG, or LH and FSH. More oocytes from large follicles (55%) completed maturation by 48 h than from small follicles (20%). Approximately 50% of oocytes remained at the GV stage at all times under all conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro maturation and fertilization of goat oocytes

Follicular cumulus-enclosed goat oocytes were matured in vitro in the presence of granulosa cells, follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol-17β. While 86% of the oocytes from follicles 2 to 6 mm in diameter achieved meiotic maturation, only 24% of the oocytes from follicles 1 to 2 mm in diameter progressed to Metaphase II. Exposure of follicle-enclosed cumulus-oocyte complexes to 20°C prior to culture resulted in 11.5% of the oocytes exhibiting abnormal meiotic spindle. This indicated that immature goat oocytes are particularly sensitive to temperature. Ejaculated spermatozoa were capacitated according to the technique previously proposed for ram sperm (1). The fertilization rates of ovulated and mechanically denuded in vitro-matured oocytes were 85 and 82.8%, respectively; 59.7% of ovulated and 57.1% of in vitro-matured oocytes were normally fertilized, as shown by the presence of both the female and the male pronucleus as well as by the remnants of the sperm tail in the ooplasm, 17 hours after insemination. Polyspermy was the main abnormality detected, and it affected almost 20% of the inseminated oocytes. The cleavage rate (two to fourcell stage) 41 hours after insemination of in vitro-matured and fertilized oocytes was 58%.

Developmental ability of in vitro matured sheep oocytes collected during the nonbreeding season and fertilized in vitro with frozen ram semen

During the nonbreeding season, oocytes recovered from ovaries of FSH-primed or nonprimed ewes were matured in the presence or absence of granulosa cells collected from ovaries of primed or nonprimed ewes prior to in vitro fertilization with either fresh or frozen-thawed sperm. Following fertilization, ova were cultured for 24 h in synthetic oviduct fluid medium (SOF) supplemented with 20% human serum at 39°C under humidified 5% CO 2, 5% O 2, 90% N 2 and then assessed for cleavage. Overall, 52% of ova cleaved. Cleavage was not affected by the source of sperm. Significantly more oocytes from primed follicles cleaved after 24 hours than those from nonprimed follicles (P<0.001). Maturation of oocytes in the presence of granulosa cells from nonprimed ewes resulted in a lower cleavage rate (44%, P<0.05) than in the presence of granulosa from primed ewes (59%) or no granulosa cells (50%). Oocytes (n = 508) from primed ewes were matured in the presence of granulosa cells (also from primed ewes) and fertilized in vitro with frozen-thawed sperm. Following in vitro culture for 24 hours, 68 of the 270 (53%) cleaved embryos were transferred to 17 recipient ewes, 15 of which remained pregnant to term, producing 24 lambs. The remaining 202 cleaved embryos were cultured for a further 5 days, of which 73 appeared to reach the morula/blastocyst stage and 61 were transferred to 16 recipients. Two ewes remained pregnant to term producing two lambs. These results demonstrate that production of sheep embryos using in vitro maturation and fertilization techniques is possible in the nonbreeding season. However, the poor viability of embryos obtained following extended culture needs to be resolved before such techniques can be usefully applied.

Production and biologic action of relaxin within the ovarian follicle: an overview

The studies described here support the concept that relaxin is a product of the ovarian follicle and interacts with systemic hormones in the local regulation of the ovary. This report reviews the work indicating that relaxin is a product of the ovarian follicle and presents evidence for the biologic action of relaxin within the follicle. Production of relaxin by cells of the theca interna was given support by immunocytochemical localization work, in vitro production studies, and detection of relaxin mRNA by in situ hybridization. The relaxin content of porcine follicular fluid was shown to increase with development induced by gonadotropins. During thecal cell culture, luteinizing hormone and porcine follicular fluid increased relaxin secretion, whereas the presence of granulosa cells was without effect. A biologic action for relaxin on connective tissue remodeling was supported by an increase in follicle-stimulating hormone-stimulated plasminogen activator activity by granulosa cells. Additional work is needed to investigate the possibility of other roles for relaxin within the follicle, to identify relaxin receptors, and to explore the interaction of relaxin with endocrine and other paracrine factors in the ovarv.