Expression, regulation, and function of progesterone receptors in bovine cumulus oocyte complexes during in vitro maturation

Progesterone (P4) exerts its effects by binding to specific genomic (nPR-A/B) and non-genomic (mPRalpha/beta, PGRMC1/2) receptors. P4 has a role in the regulation of the ovulatory cycle, but its participation in oocyte maturation in mammals has not yet been clarified. Therefore, the aim of the present study was to characterize the protein expression of P4 receptors (PRs) in bovine oocytes and cumulus cells during in vitro maturation (IVM) and to study the effect of P4 and its receptors on oocyte developmental competence. Cumulus-oocyte complexes (COCs) were subjected to IVM, in vitro fertilization, and in vitro culture. IVM was performed for 24 h in the presence or absence of P4, luteinizing hormone (LH), follicle-stimulating hormone (FSH), trilostane, promegestone (R5020), mifepristone (RU 486), or antibodies against mPRalpha or mPRbeta. Protein expression of PRs was studied by Western blotting and immunofluorescence. The results demonstrate the presence of both genomic and nongenomic PRs in bovine COCs. The dynamic changes observed in the protein expression of PRs following IVM or in response to supplementation with LH, FSH, or P4 suggest an important role during bovine oocyte maturation. Inhibition of P4 synthesis by cumulus cells or blocking of nPR and mPR alpha activity produced a decrease in bovine embryo development, indicating that P4 intracellular signaling is mediated by its interaction with nuclear and membrane PRs and is important for oocyte developmental competence.     

Changes in protein synthesis and phosphorylation patterns during bovine oocyte maturation in vitro

Sequential protein synthesis and protein phosphorylation patterns were generated by radiolabelling bovine cumulus-oocyte complexes after various periods of culture with [35S]methionine and [32P]orthophosphate respectively. The radiolabelled oocytes were assessed for their nuclear status and used individually for gel electrophoresis. Marked changes in the protein synthesis patterns were observed exclusively after germinal vesicle breakdown (GVBD), whereas oocytes which remained in the germinal vesicle stage showed a consistent protein synthesis pattern. The changes were observed after 8 and 16 h or culture, shortly after GVBD and before first polar body extrusion. From 3 h of culture, dominant phosphoprotein bands with apparent molecular weights of 24,000 and two between 50,000 and 60,000 were observed. The latter bands displayed slight molecular weight changes, which were not closely time related. After GVBD, the phosphoprotein band with Mr 19,000 was no longer observed. This study demonstrates that specific changes in protein synthesis and protein phosphorylation are programmed during bovine oocyte maturation.     

Structural aspects of bovine oocyte maturation in vitro.

Bovine cumulus-oocyte complexes (COCs) were collected from 4-8 mm follicles and graded into four categories on their morphological characteristics. These four categories were matured in vitro and processed for transmission electron microscopy at 24 h after the onset of culture. The morphology of the four groups of oocytes was analysed and compared with that of oocytes that had matured in vivo and were collected 20-23 h after the preovulatory luteinizing hormone peak. After in vivo maturation, oocytes formed a homogeneous group with respect to their morphological characteristics. After in vitro maturation, the oocytes formed a heterogeneous group with respect to their morphology between as well as within the four categories of oocytes. Oocytes from the first three categories showed the same morphology after in vitro maturation. The fourth category showed some specific characteristics: 1) vacuolization, 2) flattening of cumulus cells, and 3) almost complete lack of cortical granules in some category 4 oocytes. These characteristics are interpreted as signs of degeneration. Besides these aspects of degeneration, other deviations from normal development were seen: 1) retraction of cumulus cell process endings from the oocyte without the breaking down of these processes, 2) retardation of some aspects of the cytoplasmic maturation, and 3) incomplete cumulus expansion. It is concluded that oocytes capable of development in vitro show a large morphological variability before the onset of culture. In vitro maturation systems can support normal development, but many oocytes show signs of degeneration and deviant development after in vitro maturation.     

In vitro inhibition of oocyte nuclear maturation in the bovine

Bovine follicular oocytes (N = 5991) were exposed to an analog of cyclic adenosine 3',5'-monophosphate (cAMP), dibutyryl cyclic AMP (db-cAMP) (2.5, 5, and 10 mM), the phosphodiesterase inhibitor isobutyl methyl xanthine (IBMX, 0.2 mM), or the purine, hypoxanthine (0.5, 1.0, 2.0 mM), and the nucleoside, adenosine (0.05, 0.1, 0.2 mM), for 6 or 21 h to assess their effects on oocyte nuclear maturation. Potential effects of bovine follicular fluid (BFF) were also evaluated after different preculture washing procedures. In a separate experiment, denuded oocytes were used to study the effect of cumulus removal on meiotic inhibition. Db-cAMP decreased the frequency of germinal vesicle breakdown (GVBD) at 6 h (88% for control and 51%, 45%, and 32% for 2.5, 5, and 10 mM concentrations, respectively). IBMX had a comparable effect with only 41% of the oocytes resuming meiosis. Hypoxanthine and adenosine alone or in combinations also decreased the number of oocytes undergoing GVBD at 6 h. Only 22% GVBD occurred when the combined highest concentration of both substances was used compared to 88% in controls. If oocytes were incubated in 50% BFF after a wash in control medium during processing, 56% would resume meiosis at 6 h vs. 35% if the washing procedure included inhibitors (db-cAMP + IBMX). Total BFF (100%) during washing and maturation prevented 72% of the oocytes from resuming meiosis. Db-cAMP and IMBX combined or BFF also inhibited meiotic resumption of denuded oocytes. At 21 h, the inhibitory effects were less pronounced, with most oocytes only delayed in completing the first reduction division.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apoptosis within bovine follicular cells and its effect on oocyte development during in vitro maturation

Developmental competence of oocytes is compromised if they originate from atretic follicles. Apoptosis is the underlying process of atresia. Apoptotic changes in follicular cells are thought to influence the outcome of IVF. The aim of this study was to investigate apoptosis in different compartments of single bovine follicles (follicular wall, granulosa and cumulus cells (CC)) in relation to COC morphology, and to determine whether the addition, in vitro, of exogenous follicular cells from atretic follicles to maturing cumulus oocyte complexes (COCs) influenced the development of oocytes.Antral follicles were dissected from bovine ovaries and opened to obtain COCs and free floating granulosa cells (GC). The COCs were classified according to morphology. Apoptosis was determined in cumulus and granulosa cells and in homogenates of the remaining follicular wall.For every morphological class of COCs, a large variability of apoptotic expression was found in all follicle compartments. Follicular wall apoptosis was not correlated to COC morphology or to the percentage of apoptotic granulosa or cumulus cells. In grade 1 (best morphology) COCs, the degree of apoptosis in granulosa cells was comparable to cumulus cell apoptosis (P<0.01). The overall expression of apoptosis in granulosa cells of follicles containing grade 3 COCs (median+/-median absolute deviation: 37.8+/-13.8%) was significantly higher (P<0.05) than in follicles with grade 1 (22.7+/-10.4%) or grade 2 COCs (20.0+/-17.0%). About 48.3% of grade 3 COCs possessed strongly apoptotic cumulus cells compared to 27.8 and 28.2% of grade 1 or grade 2 COCs, respectively. Nonapoptotic cumulus complexes were observed in grades 1 and 2 COCs only.Adding exogenous follicular cells from atretic follicles to bovine COCs (grades 1 and 2) during in vitro maturation (IVM) had no impact on fertilization, blastocyst formation or hatching after IVF. This is of particular practical relevance to embryo production after ovum pick up (OPU), as during this process, good quality COCs are cultured together with simultaneously collected slightly atretic COCs.     

Cumulus cell function during bovine oocyte maturation,fertilization, and embryo development in vitro

Several contemporary micromanipulation techniques, such as sperm microinjection, nuclear transfer, and gene transfer by pronuclear injection, require removal of cumulus cells from oocytes or zygotes at various stages. In humans, the cumulus cells are often removed after 15–18 hr of sperm-oocyte coincubation to assist the identification of the fertilization status. This study was designed to evaluate the function of cumulus cells during oocyte maturation, fertilization, and in vitro development in cattle. Cumulus cells were removed before and after maturation and after fertilization for 0,7,20, and 48 hr. The cumulus-free oocytes or embryos were cultured either alone or on cumulus cell monolayers prepared on the day of maturation culture. Percentages of oocyte maturation, fertilization, and development to cleavage, morula, and blastocyst stages and to expanding or hatched blastocysts were recorded for statistical analysis by categorical data modeling (CATMOD) procedures. Cumulus cells removed before maturation significantly reduced the rate of oocyte maturation (4–26% vs. 93–96%), fertilization (0–9% vs. 91–92%), and in vitro development at all stages evaluated. Cumulus cells removed immediately prior to in vitro fertilization (IVF) or 7 hr after IVF reduced the rates of fertilization (58–60% and 71%, respectively, vs. 91–92% for controls), cleavage development (40–47% and 53–54% vs. 74–78% for controls), and morula plus blastocyst development (15% and 24% vs. 45%, P < 0.05). Cumulus cell co-culture started at various stages had no effect on fertilization and cleavage development but significantly improved rates of embryo development to morula or blastocyst stages (P < 0.05). Cumulus cell removal at 20 hr after IVF resulted in similar development to controls (P > 0.05) at all stages tested in this study. The intact state of surrounding cumulus cells of oocytes or embryos appears to be beneficial before or shortly after insemination (at or before 7 hr of IVF) but not essential at 20 hr after IVF. © 1995 Wiley-Liss, Inc.     

Melatonin supplementation during in vitro maturation of oocyte enhances subsequent development of bovine cloned embryos

Oocyte quality, which is directly related to reprogramming competence, is a major important limiting factor in animal cloning efficiency. Compared with oocytes matured in vivo, in vitro matured oocytes exhibit lower oocyte quality and reprogramming competence primarily because of their higher levels of reactive oxygen species. In this study, we investigate whether supplementing the oocyte maturation medium with melatonin, a free radical scavenger, could improve oocyte quality and reprogramming competence. We found that 10⁻⁹ M melatonin effectively alleviated oxidative stress, markedly decreased early apoptosis levels, recovered the integrity of mitochondria, ameliorated the spindle assembly and chromosome alignment in oocytes, and significantly promoted subsequent cloned embryo development in vitro. We also analyzed the effects of melatonin on epigenetic modifications in bovine oocytes. Melatonin increased the global H3K9 acetylation levels, reduced the H3K9 methylation levels, and minimally affected DNA methylation and hydroxymethylation. Genome-wide expression analysis of genes in melatonin-treated and nontreated oocytes was also conducted by high-throughput RNA sequencing. Our results indicated that melatonin ameliorates oocyte oxidative stress and improves subsequent in vitro development of bovine cloned embryos.     

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)     

Surface alterations of the bovine oocyte and its investments during and after maturation and fertilization in vitro

Surface characteristics of the bovine oocyte and its investments before, during, and after maturation, and fertilization in vitro were evaluated by scanning electron microscopy (SEM). Oocyte diameters were also measured during SEM analysis of the oocyte. The cumulus cells manifested a compact structure with minimal intercellular spaces among them in the immature oocytes. These became fully expanded with increased intercellular spaces after maturation in vitro, but contracted again after fertilization. The zona pellucida (ZP) showed a fibrous, open mesh-like structure in the maturing and matured oocytes. The size and number of meshes on the ZP decreased dramatically after fertilization. The vitelline surface of immature oocytes was characterized by distribution of tongue-shaped protrusions (TSPs) varying in density. After 10 and 22 hr of maturation incubation, oocyte surface microvilli (MV) increased to become the predominant surface structure, and TSPs decreased substantially. The vitelline surface of fertilized oocytes (at 6 and 20 hr) was similar to that of the matured oocytes, but unfertilized oocytes had less dense MV than did fertilized oocytes (at 20 hr). The diameter of the oocytes decreased from 99 to 80 μm during maturation and increased to 106 μm after insemination (P < 0.05). Membrane maturation was characterized by surface changes from a TSP-predominant pattern to a MV-predominant pattern. Thus, the bovine oocyte maturation process was found to involve the expansion of cumulus cells and the maturation of the ZP, which changes dramatically upon fertilization. Also, volumetric changes occurred in ooplasm processed for SEM following oocyte maturation and insemination. © 1994 Wiley-Liss, Inc.     

Differential regulation of histone H1 and ribosomal S6 kinases during sea star oocyte maturation

In the preceding paper [Pelech, S.L., Meijer, L., & Krebs, E.G. (1987) Biochemistry (preceding paper in this issue)], at least three activated kinases were detected in soluble extracts from sea star oocytes induced to undergo maturation by 1-methyladenine (1-MeAde). Coincident with nuclear envelope breakdown (20 min after exposure to 1-MeAde), there was a rapid activation of a histone H1 kinase that eluted from DEAE-Sephacel with a conductivity of approximately 6 mmho. By contrast, 60-min treatment of the oocytes with 1-MeAde was required for maximal activation of two kinases, each of which phosphorylated a synthetic peptide, Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (RRLSSLRA), patterned after a phosphorylation site sequence from ribosomal protein S6. These RRLSSLRA kinases were released from DEAE-Sephacel with elution conductivities of approximately 6 and approximately 10.5 mmho. The 1-MeAde dose-response curves for maturation induction and activation of the histone H1 and RRLSSLRA kinases were superimposable. Both oocyte maturation and the activation of the kinases required the presence of 1-MeAde during the hormone-dependent period. When 1-MeAde was removed after this period, full histone H1 kinase activation still occurred and maturation was induced. Forskolin pretreatment of the oocytes, by elevating the basal cAMP level more than 35-fold, doubled the hormone-dependent period and similarly delayed the onset of histone H1 kinase activation by 1-MeAde. However, postmaturation activation of the RRLSSLRA kinases was completely blocked by forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of serum and hormones on bovine oocyte maturation and fertilization in vitro

Three approaches were investigated for improvement of in vitro maturation (IVM), in vitro fertilization (IVF), and early embryonic development in cattle. These were: 1) Selection of oocytes, 2) medium supplementation with fetal calf serum (FCS) and cow sera (DO, Dl, D10, and D20 to correspond with estrus, metestrus, diestrus, and proestrus, respectively), and 3) addition of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol-17β (E₂)during maturation. Greater proportions (percentage) of oocytes initially selected for their compact cumulus cells completed IVM and IVF when compared to unselected oocytes (P < .05). Proportions (percentage) of selected oocytes that matured and cleaved after in vitro insemination according to serum type used for IVM were: FCS: 110/175 (62.9%) and 37/110 (33.6%) and DO: 130/145 (89.7%) and 52/130 (40.0%); D1 127/130 (97.7%) and 41/127 (32.3%); D10 95/98 (96.9%) and 35/95 (36.8%); D20:113/116 (97.4%) and 49/113 (43.4%). A higher proportion (P < .05) of embryos resulting from the D20 group reached four- and eight-cell stages. In FCS-supplemented maturation media with no hormones added during maturation (control), results of IVM and IVF were 157/265 (59.2%) and 39/157 (24.8%), respectively. With E₂ (1 μg/ml) and FSH (5 μg/ml), comparable results were 189/215 (87.9%) and 71/189 (37.6%); with E₂ (1 μg/ml) plus LH (10 μml), 280/327 (85.6%) and 111/280 (39.6%). Added hormones improved IVM results (P < .05) and, when FSH or LH was added with E₂, in vitro development to four- and eight-cell stages was markedly enhanced (P < .05). Selection of oocytes, D20 serum, and added E₂ and FSH or LH for IVM improved in vitro development of bovine embryos after IVF.     

Effect of C-type natriuretic peptide pretreatment on in vitro bovine oocyte maturation

C-type natriuretic peptide (CNP) has been considered as a physiological meiotic inhibitor that stimulates the cGMP production by cumulus cell natriuretic peptide receptor 2 (NPR2), which inhibits oocyte phosphodiesterase type 3 activity and increases cAMP. In this study, we explored the effect of CNP pretreatment on the in vitro maturation (IVM) of bovine oocytes by examining changes in cleavage rate, blastocyst formation, mitochondrial DNA (mtDNA) copy number, reactive oxygen species (ROS) level, glutathione (GSH) content, and redox state. Our results showed that 200 nM CNP could effectively maintain meiotic arrest of bovine oocytes in vitro within 6 h. The two-step IVM system in which oocytes were pretreated with 200 nM CNP for 6 h and then cultured IVM for 28 h yielded a significantly (P < 0.05) increased blastocyst rate and cell number after in vitro fertilization (IVF) while compared to the conventional one-step IVM method. In addition, in comparison with the conventional 24-h matured oocyte, oocytes pretreated with 200 nM CNP for 6 h followed by 28 h IVM resulted in significantly (P < 0.05) higher mtDNA copy number and ROS levels in oocytes, while GSH level significantly (P < 0.05) decreased. Remarkably, regardless of treatment, no changes were observed in FAD++, NAD(P)H autofluorescence intensity, and redox ratio (FAD++/NAD(P)H) within the oocytes, maintaining a healthy metabolic equilibrium of redox throughout the two-step IVM. In conclusion, these results indicate that CNP pretreatment could dramatically improve the quality of bovine oocytes during in vitro maturation.     

Effects of caffeine on in vivo and in vitro oocyte maturation in mice

The objective was to investigate, using a mouse model, the effects of caffeine on the number of ovulated oocytes, the rate of oocyte maturation, the susceptibility of oocytes to activating stimuli, spindle morphology, and distribution of cortical granules (CGs). Mice were given caffeine (150 mg/kg body weight ip) at various times relative to hCG (-2, 0, and +2h); in an in vitro study, 1, 5 or 10 mM caffeine was added to the maturation culture. Caffeine had no effect on the quality of oocytes in vivo maturation, but caffeine was detrimental to the quality of oocytes matured in vitro. Further studies are needed to determine caffeine concentration in follicles relative to that in culture medium.