Effect of maturation stage at cryopreservation on post-thaw cytoskeleton quality and fertilizability of equine oocytes

Oocyte cryopreservation is a potentially valuable technique for salvaging the germ-line when a valuable mare dies, but facilities for in vitro embryo production or oocyte transfer are not immediately available. This study examined the influence of maturation stage and freezing technique on the cryopreservability of equine oocytes. Cumulus oocyte complexes were frozen at the immature stage (GV) or after maturation in vitro for 30 hr (MII), using either conventional slow freezing (CF) or open pulled straw vitrification (OPS); cryoprotectant-exposed and untreated nonfrozen oocytes served as controls. After thawing, GV oocytes were matured in vitro, and MII oocytes were incubated for 0 or 6 hr, before staining to examine meiotic spindle quality by confocal microscopy. To assess fertilizability, CF MII oocytes were subjected to intracytoplasmic sperm injection (ICSI) and cultured in vitro. At 12, 24, and 48 hr after ICSI, injected oocytes were fixed to examine their progression through fertilization. Both maturation stage and freezing technique affected oocyte survival. The meiosis resumption rate was higher for OPS than CF for GV oocytes (28% vs. 1.2%; P < 0.05), but still much lower than for controls (66%). Cryopreserving oocytes at either stage induced meiotic spindle disruption (37%-67% normal spindles vs. 99% in controls; P < 0.05). Among frozen oocytes, however, spindle quality was best for oocytes frozen by CF at the MII stage and incubated for 6 hr post-thaw (67% normal); since this combination of cryopreservation/IVM yielded the highest proportion of oocytes reaching MII with a normal spindle (35% compared to <20% for other groups), it was used when examining the effects of cryopreservation on fertilizability. In this respect, the rate of normal fertilization for CF MII oocytes after ICSI was much lower than for controls (total oocyte activation rate, 26% vs. 56%; cleavage rate at 48 hr, 8% vs. 42%: P < 0.05). Thus, although IVM followed by CF yields a respectable percentage of normal-looking MII oocytes (35%), their ability to support fertilization is severely compromised.     

Effect of macromolecule supplementation during in vitro maturation of goat oocytes on developmental potential

In vitro maturation (IVM) of goat oocytes with serum-supplemented media results in oocytes with reduced developmental potential. The objective of this study was to develop a defined medium for IVM of goat oocytes that better supports subsequent embryonic development. Cumulus oocyte complexes (COC) were matured for 18-20 hr in: Experiment (1), tissue culture medium 199 (TCM199) with 10% (v/v) goat serum or modified synthetic oviduct fluid maturation medium (mSOFmat) with 2.5, 8.0, or 20.0 mg/ml bovine serum albumin (BSA); Experiment (2), mSOFmat with 4.0, 8.0, 12.0, or 16.0 mg/ml BSA; or Experiment (3), 1.0 mg/ml polyvinyl alcohol (PVA; control), 4.0 mg/ml BSA, 0.5 mg/ml hyaluronate plus 0.5 mM citrate, or hyaluronate, citrate, and BSA. Mature COC were coincubated for 20-22 hr with 12-15 x 10(6) sperm/ml in modified Brackett and Oliphant (mBO) medium. Embryos were cultured for a total of 7 days in G1/2, and evaluated for cleavage, and blastocyst development, hatching, and total cell numbers. In the first experiment, more (P < 0.05) blastocysts developed per cleaved embryo following maturation in mSOFmat with 2.5 or 8.0 mg/ml BSA than with 20.0 mg/ml BSA or TCM199 with 10% goat serum. The various concentrations of BSA used in the second experiment did not affect (P > 0.05) any of the developmental endpoints examined. In the third experiment, developmental potential of oocytes matured with PVA or hyaluronate with citrate was not different (P > 0.05) from oocytes matured in the presence of BSA. These results demonstrate that developmentally competent goat oocytes can be matured under defined conditions.     

Luteinizing hormone-enhanced in vitro maturation of bovine oocytes with and without protein supplementation

Luteinizing hormone was shown to enhance maturation of immature oocytes obtained from slaughtered cattle as reflected by elevated proportions of oocytes that fertilized and reached blastocyst stages in vitro after in vitro fertilization (IVF). Higher proportions of ova were fertilized in vitro after in vitro maturation (IVM) in modified TCM-199 (TCM-199 + BSA + LH [USDA-bLH-B-5, 100 micrograms/ml]) than in TCM-199 alone (p less than 0.01). Further improvement in IVF (p less than 0.005) followed IVM when 20% proestrous (Day 20) bovine serum replaced the BSA, but similar proportions of inseminated ova (22.2% and 22.6%) developed into blastocysts. The positive LH effect was verified in defined conditions for IVM. Exposure of oocytes to the purified LH preparation (without any other added protein or biological substances) during IVM improved IVF (39.7% in TCM-199 vs. 73.5% in TCM-199 + LH; p less than 0.001) and blastocyst development (7.9% vs. 28.2%; p less than 0.005), respectively. Efforts to better define effective concentrations of LH revealed no difference in viability after IVM with 50 micrograms LH/ml vs. 100 micrograms LH/ml (27.0% vs. 28.3%, respectively); 10 micrograms LH/ml did not enhance viability when compared to TCM-199 alone (10.8% vs. 9.9%). Results demonstrate potential utility of this approach for investigation of factors influencing mammalian development by specific effects initiated during the interval of oocyte maturation.     

Influence of insulin-like growth factor-I and its interaction with gonadotropins, estradiol, and fetal calf serum on in vitro maturation and parthenogenic development in equine oocytes

The effects of insulin-like growth factor-I (IGF-I) and its interaction with gonadotropins, estradiol, and fetal calf serum (FCS) on in vitro maturation (IVM) of equine oocytes were investigated in this study. We also examined the role of IGF-I in the presence or absence of gonadotropins, estradiol, and FCS in parthenogenic cleavage after oocyte activation with calcium ionophore combined with 6-dimethylaminopurine (6-DMAP), using cleavage rate as a measure of cytoplasmic maturation. Only equine cumulus-oocyte complexes with compact cumulus and homogenous ooplasm (n = 817) were used. In experiment 1, oocytes were cultured in TCM-199 supplemented with BSA, antibiotics, and IGF-I at 0 (control), 50, 100, 200 ng/ml, at 39 degrees C in air with 5% CO(2), 95% humidity for 36 or 48 h. In experiment 2, oocytes were cultured with FSH, LH, estradiol, and FCS with IGF-I at the concentration that promoted the highest nuclear maturation rate in experiment 1. In experiment 3, oocytes from the three experimental groups (IGF-I; hormones; and IGF-I + hormones) were chemically activated by exposure to calcium ionophore followed by culture in 6-DMAP. In experiment 1, IGF-I stimulated equine oocyte maturation in a dose-dependent manner with the highest nuclear maturation rate at a concentration of 200 ng/ml. No significant effect of IGF-I on nuclear maturation was observed in experiment 2. In experiment 3, a significant difference in cleavage rate was observed between the hormone + IGF-I group (15 of 33; 45.4%) compared with IGF-I (10 of 36; 27.8%) and hormone (4 of 31; 12.9%) alone (P < 0.05). These results demonstrated that IGF-I has a positive effect on nuclear maturation rate of equine oocytes in vitro. The addition of IGF-I to an IVM medium containing hormones and FCS did not increase nuclear maturation, but resulted in a positive effect on cytoplasmic maturation of equine oocytes measured by parthenogenic cleavage.     

The endocannabinoid system modulates the ovarian physiology and its activation can improve in vitro oocyte maturation

The cannabinoid (CB) system has been involved in many aspects of reproduction and it is known that the systemic chronic use of exogenous CBs are deleterious to reproductive processes. Even so, it is not known what happens in relation to the physiology of the ovary when CB receptors are absent. The present study investigated the effect of the lack of CB1 and CB2 receptors in mice ovarian morphology, folliculogenesis, oocyte retrieval, and oocyte maturation and evaluated the use of Δ9-tetrahydrocannabinol (THC) on oocyte in vitro maturation (IVM) by comparing classical IVM and two-step IVM by analyzing the meiotic competence of the oocytes and their evolution toward embryos. Thus, when CB1 and CB2 receptors were missed, the ovary area and volume was significantly less and the action of the equine chorionic gonadotropin (eCG) hormone was diminished. In addition, the mutant genotypes had fewer ovarian follicles and they were less competent after eCG administration compared with wild-type mice, and this lack of CB receptors showed a mismatch of oocyte maturation. However, the in vitro use of THC showed improvements in oocytes IVM after a Pre-IVM step for 48 hr, as those oocytes reached a significantly higher polar body rate, a larger diameter and the best result on blastocysts rate was achieved when THC was used during the IVM step.     

Thyroid stimulating hormone enhancement of bovine oocyte maturation in vitro.

Efforts to improve bovine embryonic development in vitro involved study of effects of thyroid stimulating hormone (TSH) alone or in combination with LH on bovine oocyte maturation (IVM). Putative effects were assessed by observing cumulus expansion (CE), fertilization (IVF), and development to morulae/blastocysts (M/B). Effects of prolactin (PRL) were also investigated. Variables for the 24-hr IVM interval were no hormone (control), TSH (0.1, 0.5, or 1.0 micrograms/ml) or PRL (10, 100, or 1000 micrograms/ml), luteinizing hormone (LH) (0, 10, or 100 micrograms/ml) + TSH (0.1 or 0.5 micrograms/ml), and serum (20%, v/v) + 0.5 micrograms TSH/ml; data were from 4-5 trials for each IVM treatment. Higher proportions of oocytes exhibited complete CE with hormones or serum than without (P less than 0.05). All oocytes (with and without CE) were inseminated with heparin-capacitated sperm. A higher proportion of inseminated oocytes cleaved after IVM with 0.5 micrograms TSH/ml (53.4%) than for other TSH treatments (P less than 0.05). The combination of TSH (0.1 and 0.5 micrograms/ml) with 10 micrograms LH/ml for IVM enabled higher proportions (P less than 0.05) of ova to fertilize (67.4 and 69.2%) than did medium alone (28.3%), LH (10 micrograms/ml) alone (54.1%) or serum + 0.5 micrograms TSH/ml (55.6%). No improvement in proportions undergoing fertilization was seen after addition of TSH to 100 micrograms LH/ml for IVM. Frequency of CE and cleavage did not differ among PRL treatments. More M/B developed from cleaved ova after IVM with LH or TSH than with PRL or no hormone (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth hormone and gene expression of in vitro‐matured rhesus macaque oocytes

Growth hormone (GH) in rhesus macaque in vitro oocyte maturation (IVM) has been shown to increase cumulus expansion and development of embryos to the 9–16 cell stage in response to 100 ng/ml recombinant human GH (r‐hGH) supplementation during IVM. Although developmental endpoints for metaphase II (MII) oocytes and embryos are limited in the macaque, gene expression analysis can provide a mechanism to explore GH action on IVM. In addition, gene expression analysis may allow molecular events associated with improved cytoplasmic maturation to be detected. In this study, gene expression of specific mRNAs in MII oocytes and cumulus cells that have or have not been exposed to r‐hGH during IVM was compared. In addition, mRNA expression was compared between in vitro and in vivo‐matured metaphase II (MII) oocytes and germinal vesicle (GV)‐stage oocytes. Only 2 of 17 genes, insulin‐like growth factor 2 (IGF2) and steroidogenic acute regulator (STAR), showed increased mRNA expression in MII oocytes from the 100 ng/ml r‐hGH treatment group compared with other IVM treatment groups, implicating insulin‐like growth factor (IGF) and steroidogenesis pathways in the oocyte response to GH. The importance of IGF2 is notable, as expression of IGF1 was not detected in macaque GV‐stage or MII oocytes or cumulus cells. Mol. Reprod. Dev. 77: 353–362, 2010. © 2009 Wiley‐Liss, Inc.     

Epidermal growth factor enhances meiotic resumption of canine oocytes in the presence of BSA

Despite many attempts to improve the in vitro maturation (IVM) of canine oocytes using various culture conditions, the efficiency of canine IVM remains very low compared with that of other domestic animals. In the present study we examined the effect of ovarian estrus stage on oocyte quality, and the effect of epidermal growth factor (EGF) in the presence and absence of macromolecules on the IVM of canine oocytes. More oocytes >or=100 microm in diameter were obtained from follicular ovaries than from ovaries at other estrus stages. After 72 h of culture, significantly more oocytes recovered from follicular ovaries than from anestrous and luteal ovaries were in germinal vesicle break down (GVBD). Bovine serum albumin (BSA) or fetal bovine serum (FBS) supplementation improved meiotic resumption as compared to polyvinyl alcohol (PVA) supplementation; however, there was no difference between the BSA and FBS supplements. The oocytes matured in North Carolina State University (NCSU) 37 medium containing 0.4% BSA and 100 ng/ml EGF showed the highest rates of development to the metaphase II (MII) stage when compared with the control treatment (P < 0.05). These results suggest that the estrous cycle of bitches influences the meiotic resumption of oocytes cultured in vitro, and EGF increases the meiotic resumption of canine oocytes in the presence of BSA in vitro.     

Isolation, identification and characterization of secretory proteins of IVMFC embryos and blood circulation of estrus and early pregnant goat

The aim of the present study was to isolate, identify and characterize the secretory proteins of IVM oocytes and IVMFC embryos to evaluate its immunogenecity. and identify of such proteins if any, in blood circulation of estrus and early pregnant goats. Oocytes were matured in TCM-199 with 1 microg/ml, estradiol-17beta; 0.5 microg/ml, FSH; 100 IU/ml, LH and 10% FCS on granulosa cell monolayer. After 18 hr of maturation, oocytes were further cultured in maturation medium containing 3 mg/ml polyvinyl alcohol (PVA) without serum and BSA for 12 hr and medium was collected. The IVF embryos of 4-8 cell stage were cultured in medium containing PVA without serum and BSA. Embryo culture medium was collected after 24 hr of culture and was pooled. The proteins were analyzed on SDS-PAGE (12.5%). Four secretory proteins of oocytes with approximately molecular weight of 45, 55, 65 and 95 kDa and three secretory proteins of embryos 45, 55 and 65 kDa were obtained on SDS-PAGE in silver staining. The protein profile of midluteal, estrus and early pregnant goat serum was similar and no variation was observed among the proteins on SDS-PAGE. Two secretory proteins of 55 and 65 kDa of both IVM oocytes and IVMFC embryos were observed on Western analysis. None of such proteins was observed in midluteal, estrus and early pregnant goat serum on western blotting. It can be concluded that IVM oocytes and IVMFC embryos secrete proteins in medium and two of them can develop antibody. The proteins secreted from embryos till morula stage was similar to that of oocytes. None of these oocyte/embryo released proteins were observed in blood circulation of estrus and early pregnant goats.     

In vitro acute exposure to DEHP affects oocyte meiotic maturation, energy and oxidative stress parameters in a large animal model

Phthalates are ubiquitous environmental contaminants because of their use in plastics and other common consumer products. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate and it impairs fertility by acting as an endocrine disruptor. The aim of the present study was to analyze the effects of in vitro acute exposure to DEHP on oocyte maturation, energy and oxidative status in the horse, a large animal model. Cumulus cell (CC) apoptosis and oxidative status were also investigated. Cumulus-oocyte complexes from the ovaries of slaughtered mares were cultured in vitro in presence of 0.12, 12 and 1200 μM DEHP. After in vitro maturation (IVM), CCs were removed and evaluated for apoptosis (cytological assessment and TUNEL) and intracellular reactive oxygen species (ROS) levels. Oocytes were evaluated for nuclear chromatin configuration. Matured (Metaphase II stage; MII) oocytes were further evaluated for cytoplasmic energy and oxidative parameters. DEHP significantly inhibited oocyte maturation when added at low doses (0.12 μM; P<0.05). This effect was related to increased CC apoptosis (P<0.001) and reduced ROS levels (P<0.0001). At higher doses (12 and 1200 μM), DEHP induced apoptosis (P<0.0001) and ROS increase (P<0.0001) in CCs without affecting oocyte maturation. In DEHP-exposed MII oocytes, mitochondrial distribution patterns, apparent energy status (MitoTracker fluorescence intensity), intracellular ROS localization and levels, mt/ROS colocalization and total SOD activity did not vary, whereas increased ATP content (P<0.05), possibly of glycolytic origin, was found. Co-treatment with N-Acetyl-Cysteine reversed apoptosis and efficiently scavenged excessive ROS in DEHP-treated CCs without enhancing oocyte maturation. In conclusion, acute in vitro exposure to DEHP inhibits equine oocyte maturation without altering ooplasmic energy and oxidative stress parameters in matured oocytes which retain the potential to be fertilized and develop into embryos even though further studies are necessary to confirm this possibility.     

Germinal vesicle chromatin configuration and meiotic competence is related to the oocyte source in canine

This study investigated the effect of deriving oocytes from different stages of the estrous cycle on oocyte diameter, germinal vesicle (GV) chromatin configuration, and in vitro meiotic competence in canine oocytes. Cumulus oocyte complexes (COCs) were recovered from both ovaries during anestrous, follicular, and luteal phases and in vivo ovulated oocytes. The diameter of canine oocyte was compared with or without the zona pellucida (ZP) before in vitro maturation (IVM). Also, GV chromatin configuration was evaluated before (0 h) or 72 h after IVM by fixation with 3.7% formaldehyde supplemented with 10 microg/ml Hoechst 33342 for 30 min. COCs were matured in TCM199 supplemented with 10% fetal bovine serum (FBS), 0.6 mM cysteine, 0.2 mM pyruvic acid, 50 microg/ml gentamycin sulfate, and 20 microg/ml 17beta-estradiol (E(2)) at 39 degrees C and 5% CO(2) in air for 72 h. The diameter of in vivo ovulated oocytes with the ZP (167.5+/-12.7 microm) or without ZP (133.9+/-5.3 microm) was significantly greater (p<0.05) than those of anestrous, follicular, and luteal oocytes (with ZP, 151.2+/-7.4, 153.1+/-8.8 and 152.8+/-5.4 microm, respectively; without ZP, 115.3+/-7.6, 122.1+/-4.9 and 114.3+/-6.6 microm, respectively). At 0 h, the GV-II configuration was more prevalent in oocytes from anestrual ovaries than from follicular or luteal ovaries or in vivo ovulated oocytes (63.6% versus 14.8%, 33.0%, and 0.0%; p<0.05), whereas the proportion of oocytes with the GV-V configuration was higher in follicular phase and ovulated oocytes than in oocytes from anestrus and luteal phase (57.4% and 100% versus 2.0% and 22.7%; p<0.05). However, oocytes in luteal phase exhibited diverse GV configurations (10.3%, 33.0%, 16.5%, 13.4%, and 22.7% in GV-I, GV-II, GV-III, GV-IV, and GV-V, respectively). After 72 h post-IVM, a greater percentage of in vivo ovulated oocytes progressed to MII than those oocytes collected during anestrous, follicular, and luteal phases (50.0% versus 5.5%, 11.5%, and 9.1%; p<0.05). In conclusion, the oocyte diameter, GV chromatin configuration, and meiotic maturation of canine COCs are related to the oocyte source. These results indicated that the oocyte source could be critical to nuclear progression to MII stage in canines.     

The effect of growth hormone (GH) and insulin-like growth factor-I (IGF-I) on in vitro maturation of equine oocytes

Summary The objective of this study was to test the hypothesis that equine growth hormone (eGH), in combination with insulin growth factor-I (IGF-I), influences positively in vitro nuclear and cytoplasmic maturation of equine oocytes. Cumulus-oocyte complexes were recovered from follicles that were < 25 mm in diameter, characterized by morphology and were allocated randomly as follow: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 400 ng/ml anti-IGF-I antibody. Oocytes were matured for 30 h at 38.5°C in air with 5% CO2 and then stained with 10 μg/ml propidium iodide (PI) to evaluate nuclear status and 10 μg/ml Lens culinaris agglutinin-fluorescein complex (FITC-LCA) to assess cortical granule migration by confocal microscopy. The proportion of immature oocytes that developed to the metaphase II (MII) stage in the eGH + IGF-I group (15 of 45) was greater than in the groups that were treated only with IGF-I (7 of 36, p = 0.03). Oocytes that reached MII in the control group (20 of 56; 35.7%) showed a tendency to be different when compared with eGH + IGF-I group (15 of 45; 33.3%, p = 0.08). The treated group that contained anti-IGF-I (15 of 33; 45.4%) decreased the number of oocytes reaching any stage of development when compared with eGH (47 of 72; 65.3%) and eGH + IGF-I (33 of 45; 73.3%) groups (p = 0.05) when data from MI and MII were combined. We concluded that the addition of eGH to in vitro maturation (IVM) medium influenced the in vitro nuclear and cytoplasmic maturation of equine oocytes. The use of GH and IGF-I in vitro may represent a potential alternative for IVM of equine oocytes.     

In vitro maturation, fertilization and early cleavage rates of bovine fetal oocytes

The in vitro developmental competence of oocytes harvested from 3 to 6 mm follicles from ovaries of 7.5 months to term fetuses and adult cows was compared. Cumulus oocyte complexes (COCs) were washed and placed in 200 microl droplets of maturation medium 199, supplemented with 10 microg/ml FSH, 10 microg/ml LH, 1.5 microg/ml estradiol, 75 microg/ml streptomycin, 100 IU/ml penicillin, 10 mM Hepes, and 10% fetal bovine serum (FBS) under oil and incubated for 24 h at 39 degrees C and 5% CO2. Matured oocytes were exposed to frozen-thawed TALP swim-up, heparin-capacitated sperm (20 h, 39 degrees C, 5% CO2). Presumptive zygotes were cultured in medium 199 containing 8 mg/ml BSA-V, 100 IU/ml penicillin G, 75 microg/ml streptomycin, and 10 mM Hepes (48 h, 39 degrees C, 5% CO2). Oocytes/embryos were fixed, stained with DAPI, and evaluated under fluorescent microscopy to assess maturation, fertilization, and subsequent embryonic development. There was a difference (P<0.05) between fetal and adult cow oocytes for in vitro maturation (IVM; 80.1% versus 92.0%), fertilization (69.3% versus 79.9%), and cleavage rates (36.7% versus 49.9%), respectively. Poor IVM, fertilization and embryonic development of fetal oocytes may be due to a higher incidence of blockage at germinal vesicle (GV) and metaphase-I (M-I) stage after IVM (12.0% versus 2.3% for fetal versus adult oocytes, respectively, P<0.05). Although the IVF results with fetal oocytes are poorer than with adult cow oocytes, they were still high enough to be considered for use in research and when death of the dam and/or fetus is pre-mature or sudden.     

Equine luteinizing hormone possesses follicle-stimulating hormone activity in hypophysectomized female rats

The ability of equine luteinizing hormone (eLH) to promote follicular growth and maturation in hypophysectomized rats has been assessed. A single injection of equine LH has been shown to promote the growth of a large number of antral and preovulatory follicles. In addition, equine LH markedly increased serum estrogen levels and uterine weight. Furthermore, equine LH, like equine chorionic gonadotropin (eCG; PMSG) was able to significantly enhance the incorporation of [3H]thymidine into ovarian DNA, an activity shown to be specific to hormones having follicle-stimulating hormone (FSH) activity. Equine LH treated with an FSH antibody immunoaffinity column to remove any possible contamination still exhibited the above activity, demonstrating that the FSH activity is intrinsic to the eLH molecule. Equine LH has also been shown to be capable of inducing LH receptors in granulosa cells of ovaries of hypophysectomized rats, an activity specific to FSH-like hormones. From the doses required of eLH and the degree of response observed, it is concluded, however, that eLH in the hypophysectomized rat is less active than eCG as an FSH.     

Follicle-stimulating hormone and growth hormone act differently on nuclear maturation while both enhance developmental competence of in vitro matured bovine oocytes

This study was designed to investigate the effect of follicle-stimulating hormone (FSH) on nuclear maturation, fertilization, and early embryonic development of in-vitro-matured bovine oocytes and to find out whether this effect is exerted through a cyclic adenosine monophosphate (cAMP) signal transduction pathway. In addition the effect of the combination of FSH and growth hormone (GH) on subsequent cleavage and embryo development was studied. Therefore cumulus oocyte complexes were cultured in the presence of FSH (0.05 IU/ml) and the nuclear stage of the oocytes was assessed using 4,6-diamino-2-phenyl-indole (DAPI) staining either after 16, 20, or 24 hr of in vitro maturation or 18 hr after the onset of fertilization. To assess the effect of FSH and the combination of FSH and GH added during in vitro maturation on the developmental capacity of the oocytes, cumulus oocyte complexes were incubated in the presence of either FSH (0.05 IU/ml) or FSH (0.05 IU/ml) plus GH (100 ng/ml) for 22 hr, followed by in vitro fertilization and in vitro embryo culture. To investigate whether FSH-induced oocyte maturation is exerted through the cAMP pathway, cumulus oocyte complexes were cultured in M199 supplemented with FSH (0.05 IU/ml) and H-89 (10 μM), a specific inhibitor of cAMP-dependent protein kinase A. After 16 hr of culture, the proportion of oocytes in metaphase II (MII) stage was determined. Cultures with GH and without FSH and H-89 served as controls. The percentage of MII oocytes at 16 hr of incubation was significantly lower (P < 0.001) in the presence of FSH than in the control group, while the number of MII oocytes beyond 20 hr did not differ from the control group. That points to a transient inhibition of nuclear maturation by FSH. Opposite to FSH, addition of GH during in vitro maturation significantly enhanced the number of MII oocytes after 16 hr of culture (P < 0.001), which points to the acceleration of nuclear maturation by GH. Addition of FSH during in vitro maturation significantly enhanced the proportion of normal fertilized oocytes, cleaved embryos and blastocysts (P < 0.001). Similarly, addition of GH during in vitro maturation significantly enhanced the number of cleaved embryos and blastocysts (P < 0.001); however, in vitro maturation in the presence of GH and FSH did not result in an extra enhancement of the embryo development. Both the inhibition of nuclear maturation by FSH and its acceleration by GH was completely abolished by H-89. In conclusion, in vitro maturation of bovine oocytes in the presence of FSH retards nuclear maturation via a cAMP-mediated pathway, while it enhances fertilizability and developmental ability of the oocytes. Supplementation of GH and FSH during in vitro maturation did not result in an extra increase in the number of blastocysts following in vitro fertilization and in vitro embryo culture. Mol. Reprod. Dev. 51:339–345, 1998. © 1998 Wiley-Liss, Inc.     

Prematuration of bovine oocytes with butyrolactone I: effects on meiosis progression, cytoskeleton, organelle distribution and embryo development

The effects of prematuration (PM) of bovine oocytes with butyrolactone I (BLI) for 24h on meiosis progression, cell structures and embryo development were assessed. Germinal vesicle (GV) rates decreased (97.4-65.1%, P<0.05) with decreasing BLI concentrations (100-25muM). Without BSA in PM medium, GV rates were similar (98.7-97.2, P>0.05) with low BLI (10-25muM). After in vitro maturation (IVM) for 24h, metaphase II (MII) rates for controls (IVM only) were similar (91.1%, P>0.05) to PM with 10muM BLI in BSA-free medium (B10=91.5%) and 100muM BLI in medium with BSA (B100=92.4%). Meiosis resumption occurred earlier in treated oocytes (71.4-74.3% in GV for B10 and B100, respectively, after 6h IVM compared with 97.3% in controls, P<0.05). By 18h of IVM, most oocytes reached MII (72.0-78.9%, P>0.05). Microtubules and microfilaments were unaffected by BLI. Cortical granules (CG) migration was reversibly blocked by BLI. Mitochondria translocation was partially blocked by PM culture and after IVM more oocytes in B10 and B100 (95.2 and 98.2%, respectively) had mitochondria translocated to a mature pattern (all cytoplasm) than controls (81.5%, P<0.05). Cleavage rates were similar (81-87%, P>0.05), but blastocysts (day 7) decreased in B100 (33.0%, P<0.05) compared with controls and B10 (38.3 and 41.6%, respectively). Day 8 hatching rates (11.0-19.2%) and mean total cell numbers (136-150) were similar (P>0.05). PM did not improve oocyte competence but also did not cause major structural alterations, suggesting that PM may be improved and used to study the mechanisms involved in oocyte differentiation.     

Capacity of adult and prepubertal mouse oocytes to undergo embryo development in the presence of cysteamine

The present study was carried out to study de novo glutathione (GSH) synthesis and to evaluate the effect of stimulating GSH synthesis during in vitro maturation (IVM) of adult and prepubertal mouse oocytes on the embryo developmental rate. Adult (8 weeks old) and prepubertal mice (24-26 days old) were primed with 5 IU of PMSG and oocytes were retrieved from the ovary 48 hr later for IVM. After IVM (18 hr) Cumulus oocyte complexes (COC) were in vitro fertilized (IVF) and in vitro culture (IVC) in order to observe embryo development. The IVM medium was supplemented with: 0, 25, 50, 100, or 200 microM of cysteamine. To study the novo GSH synthesis, 5 mM BSO was added during IVM of adult or prepubertal oocyte. Developmental rates up to blastocyst were recorded for each group. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and HCG. After IVM of adult mice oocytes, an improvement was observed on embryo development in all the supplemented groups when compared with the untreated group (P < 0.05). No differences were observed in blastocyst rate among IVM oocytes with cysteamine and ovulated oocytes. Prepubertal IVM mouse oocytes had a lower cleavage rate compared with ovulated oocytes (P < 0.05). Cysteamine failed to improve prepubertal oocytes developmental rates (P > 0,05). 2-cell embryos, coming from IVM prepubertal oocytes and ovulated oocytes had the same preimplantation developmental rate up to the blastocyst stage. In prepubertal, and adult oocytes an inhibition of embryo development was observed when buthionine sulfoximide (BSO), a specific inhibitor of the gamma-glutamylcysteine synthetase, was added during oocyte maturation (P < 0.01). In conclusion, an improvement in mouse embryo development was observed when cysteamine was added to the IVM medium of adult mice oocytes. In prepubertal oocytes cysteamine addition during oocyte maturation failed to improve embryo developmental rates. The presence of BSO lowered or completely blocked blastocyst development. This proves that, de novo GSH synthesis during oocyte maturation of adult and prepubertal oocytes undoubtedly plays an important role in embryo development. The improvement on oocyte competence observed in adult mice oocytes is probably related to intracellular GSH synthesis stimulated by cysteamine. Nevertheless the reason why cysteamine failed to improve prepubertal oocytes competence remains as an open question.     

The meiotic response of cumulus cell-enclosed mouse oocytes to follicle-stimulating hormone in the presence of different macromolecules.

Experiments were carried out to determine the effect of different macromolecules on the follicle-stimulating hormone (FSH)-induced maturation of mouse oocytes in culture. Cumulus cell-enclosed oocytes (CEO) were isolated from gonadotropin-primed mice and maintained in meiotic arrest for 17-18 h with the cAMP analogue, dibutyryl cAMP (dbcAMP). Germinal vesicle breakdown (GVB) was stimulated by the addition of FSH. Medium was supplemented with either no macromolecule or with varying concentrations of polyvinylpyrrolidone (PVP), polyvinylalcohol (PVA), crystallized bovine serum albumin (BSA), or fetal bovine serum (FBS). Oocyte maturation in all FSH-free cultures occurred at a frequency of about 30% or below. High frequencies of maturation were achieved when FSH was added to macromolecule-free medium or to cultures containing PVP, PVA, or BSA. Crystallized BSA was the most effective of these in supporting stimulation of maturation (94% GVB at 3 mg/ml, compared with 72-74% with synthetic polymer-supplemented or macromolecule-free media). The BSA effect was not due to contaminating fatty acids, and a less pure fraction V BSA was not as effective in supporting FSH-induced maturation. FBS suppressed FSH stimulation of maturation in a dose-dependent fashion. Sera from pigs, goats, horses, and rats were also inhibitory, but bovine calf serum (BCS) permitted a high maturation frequency (80% GVB). When added to medium containing either FBS or BCS, crystallized BSA had no effect on FSH-stimulated maturation, but fraction V BSA suppressed maturation in both serum-supplemented media. Under no conditions did FSH stimulate maturation in cumulus cell-free oocytes. These results demonstrate that hormone-induced oocyte maturation is supported in vitro by nonprotein polymers as well as BSA and that the behavior of the oocyte-cumulus cell complex depends on the purity of the BSA sample. In addition, serum contains inhibitory factors that suppress the positive response to FSH. Thus, the choice of macromolecular supplement is of critical importance when testing the hormone responsiveness of isolated cumulus cell-enclosed oocytes in culture.     

Influence of equine growth hormone,insulin-like growth factor-I and its interaction with gonadotropins on in vitro maturation and cytoskeleton morphology in equine oocytes

In horses, successful in vitro fertilization procedures are limited by our inability to consistently mature equine oocytes by in vitro methods. Growth hormone (GH) is an important regulator of female reproduction in mammals, playing an important role in ovarian function, follicular growth and steroidogenesis. The objectives of this research were to investigate: the effects of equine growth hormone (eGH) and insulin-like growth factor-I (IGF-I) on the in vitro maturation (IVM) of equine oocytes, and the effects of eGH in addition to estradiol (E₂), gonadotropins (FSH and LH) and fetal calf serum (FCS) on IVM. We also evaluated the cytoskeleton organization of equine oocytes after IVM with eGH. Equine oocytes were aspirated from follicles <30 mm in diameter and matured for 30 h at 38.5°C in air with 5% CO₂. In experiment 1, selected cumulus–oocyte complexes (COCs) were randomly allocated as follows: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 200 ng/ml anti-IGF-I. In addition to these treatment groups, we also added 1 μg/ml E₂, 5 IU/ml FSH, 10 IU/ml LH and 10% FCS in vitro (experiment 2). Oocytes were stained with markers for microtubules (anti-α-tubulin antibody), microfilaments (AlexaFluor 488 Phalloidin) and chromatin (TO-PRO₃-iodide) and assessed via confocal microscopy. No difference was observed when eGH and IGF-I was added into our IVM system. However, following incubation with eGH alone (40%) and eGH, E₂, gonadotropins and FCS (36.6%) oocytes were classified as mature v. 17.6% of oocytes in the control group (P < 0.05). Matured equine oocytes showed that a thin network of filaments concentrated within the oocyte cortex and microtubules at the metaphase spindle showed a symmetrical barrel-shaped structure, with chromosomes aligned along its midline. We conclude that the use of E₂, gonadotropins and FCS in the presence of eGH increases the number of oocytes reaching oocyte competence.