Effect of dominant follicle persistence on follicular fluid oestradiol and inhibin and on oocyte maturation in heifers

The aim of the present study was to characterize in detail the cytoplasmic and nuclear morphology of cattle oocytes recovered from follicles that are dominant for more than 9 days (with low fertility after ovulation), and to relate morphological changes to intrafollicular markers of follicle health. Beef heifers received prostaglandin F2 alpha and a synthetic progestagen (3 mg Norgestomet) for 2 or 10 days on the first day of dominance of the second dominant follicle (DF2) of the oestrous cycle, to give a 4 day (n = 19; N2) or 12 day (n = 21; N10) duration of dominance of the dominant follicle at ovariectomy 18 h after implant removal and before the predicted gonadotrophin surge. Ultrasound scanning determined emergence of a new wave of follicles in five N10 heifers the day before (n = 1) or day of ovariectomy (n = 4) (N10-NonDom). Dominant follicles from the remaining N10 heifers (N10-Dom) were larger (P < 0.05) on the day of ovariectomy (17.8 +/- 0.6 mm) than those from N2 heifers (13.6 +/- 0.4 mm). The oestradiol:progesterone ratio of follicular fluid from N10-Dom heifers was reduced (21.7 +/- 3.1 versus 34.1 +/- 4.4; P < 0.05), while inhibin A (as measured by immunoradiometric assay) was increased (12.7 +/- 1.0 versus 9.0 +/- 0.7 micrograms ml-1; P < 0.05) compared with N2 heifers. Eleven of twelve N2 oocytes demonstrated nuclear activation without germinal vesicle breakdown, while seven of eight N10-Dom oocytes had undergone germinal vesicle breakdown and had progressed to metaphase I (6/8) or II (1/8). In contrast to N2 oocytes, N10-Dom oocytes showed a larger perivitelline space containing more cumulus cell process endings, vacuoles, irregular vesicles, and more mitochrondia and lipid droplets throughout the ooplasm, yet the degree of cumulus cell expansion and atresia was similar. Thus, final oocyte maturation leading to metaphase I is initiated in most dominant follicles with a dominance period of > 9 days before the gonadotrophin surge and is associated with a reduction in dominant follicle health. However, ovulatory ability is maintained and will lead to the ovulation of aged oocytes, markedly reducing subsequent pregnancy rates.     

Aspects of cellular proliferation during follicular atresia in the dog ovary

Summary Autoradiography after pulse labelling with [³H] thymidine was applied to investigate the proliferation processes in the granulosa and theca related to follicular atresia of the dog ovary during metestrus.The number of proliferating cells depends on the follicle type and its atretic stage. There is less proliferation in smaller secondary follicles than either in larger ones or tertiary follicles. While in early atresia tertiary follicles show the highest labelling indices, in advanced atresia the larger secondary follicles are those with the highest values. For each follicle type a decline in the labelling indices can be observed from early to terminal atresia. Tertiary follicles show a precipitous decrease in the labelling index between early and advanced atresia. There is a continuous gradient of proliferation from the center of the follicle over the peripheral granulosa to the theca. In tertiary follicles, an inverse correlation between labelling and necrosis of granulosa cells can be observed.     

HDAC3 inhibition disrupts the assembly of meiotic apparatus during porcine oocyte maturation

Histone deacetylases (HDACs) are involved in a wide array of biological processes. However, the role of HDAC3 in porcine oocytes remains unclear. In the current study, we examine the effects of HDAC3 inhibition on porcine oocyte maturation using RGFP966, a selective HDAC3 inhibitor. We find that suppression of HDAC3 activity prevents not only the expansion of cumulus cells but also the meiotic progression of oocytes. It is interesting to note that HDAC3 displays a spindle-like distribution pattern as the porcine oocytes enter meiosis. In line with this, confocal microscopy reveals the high frequency of spindle defects and chromosomal congression failure in metaphase oocytes exposed to RGFP966. Moreover, HDAC3 inhibition results in the hyperacetylation of α-tubulin during oocyte meiosis. These findings indicate that HDAC3 activity might control the microtubule stability via the deacetylation of tubulin, which is critical for maintaining the proper spindle assembly, accurate chromosome separation, and orderly meiotic progression during porcine oocyte maturation.     

Aromatase is expressed and active in the rainbow trout oocyte during final oocyte maturation

While it is generally well accepted that the ovarian follicular sites of estradiol-17β (E2) synthesis are restricted to somatic cells, the possible contribution of the germinal compartment has received little or no attention in teleosts. In order to demonstrate the expression of ovarian aromatase in the oocyte, cyp19a1a mRNA was studied in ovarian follicles by in situ hybridization. In addition, the expression of cyp19a1a was studied in both somatic and germinal compartments of the ovarian follicle in rainbow trout (Oncorhynchus mykiss) during final oocyte maturation (i.e., maturational competence acquisition and subsequent meiosis resumption) by real-time PCR. The enzymatic activity of ovarian aromatase was also studied in both somatic and germinal compartments of the ovarian follicle. Finally, E2 levels were monitored in follicle-enclosed oocytes throughout the pre-ovulatory period. We were able to demonstrate a significant ovarian aromatase expression and activity in the late vitellogenic oocyte. Furthermore, a dramatic decrease in aromatase expression and activity occurs in the oocyte during late oogenesis, concomitantly with the trend observed in surrounding follicular layers. We also report an unexpected increase of E2 levels in the oocyte during the pre-ovulatory period. To our knowledge, these observations are reported for the first time in any teleost species. Together, our data support the hypothesis of the participation of the germinal compartment in follicular estrogen synthesis and a biological role of E2 during oocyte and/or early embryo development.     

Nuf2 is required for chromosome segregation during mouse oocyte meiotic maturation

Nuf2 plays an important role in kinetochore-microtubule attachment and thus is involved in regulation of the spindle assembly checkpoint in mitosis. In this study, we examined the localization and function of Nuf2 during mouse oocyte meiotic maturation. Myc₆-Nuf2 mRNA injection and immunofluorescent staining showed that Nuf2 localized to kinetochores from germinal vesicle breakdown to metaphase I stages, while it disappeared from the kinetochores at the anaphase I stage, but relocated to kinetochores at the MII stage. Overexpression of Nuf2 caused defective spindles, misaligned chromosomes, and activated spindle assembly checkpoint, and thus inhibited chromosome segregation and metaphase-anaphase transition in oocyte meiosis. Conversely, precocious polar body extrusion was observed in the presence of misaligned chromosomes and abnormal spindle formation in Nuf2 knock-down oocytes, causing aneuploidy. Our data suggest that Nuf2 is a critical regulator of meiotic cell cycle progression in mammalian oocytes.     

Effects of oxygen tension and follicle cells on maturation and fertilization of porcine oocytes during in vitro culture in follicular fluid

The objective was to investigate the effects of oxygen tension and follicle cells (FCs) during in vitro maturation of porcine oocytes in only porcine (Sus scrofa domesticus) follicular fluid (pFF), using static and non-static (rotating) culture systems, on the nuclear maturation and subsequent in vitro fertilization of the oocytes. In the first experiment, cumulus-oocyte complexes (COCs) were matured for 48 h in pFF supplemented with (+) or without (−) FCs (5.2 × 10⁶ cells/mL), using the static (S) and rotating (R) culture systems (+FC/S, −FC/S, +FC/R, and −FC/R) under 5% or 20% O₂. Co-culture with FCs in the static culture system (+FC/S) had a detrimental effect on the meiotic competence of oocytes, whereas co-culture with FCs in the rotating culture system (+FC/R) increased maturation rates. In both culture systems, oxygen tension had no apparent effects on meiotic competence of oocytes, irrespective of culture system and FC addition. In the second experiment, COCs were matured under 5% or 20% O₂ using the −FC/S or +FC/R culture systems and then fertilized. Oxygen tension had no significant effects on fertilization parameters, irrespective of the culture system. The rotating culture system increased rates of sperm penetration and male pronuclear formation and decreased polyspermic fertilization compared with the static culture system (P < 0.05). In conclusion, both −FC/S and +FC/R culture systems supported meiotic competence, irrespective of oxygen tension. However, the +FC/R culture system may be superior to the −FC/S culture system for promoting fertilization.     

Oocyte maturation inhibitor, inhibin and steroid concentrations in porcine follicular fluid at various stages of the oestrous cycle

Oocyte maturation inhibitor (OMI), inhibin, progesterone and oestradiol 17 beta concentrations were measured in fluid collected from small (less than 3 mm), medium size (3-6 mm) and large (greater than 6 mm) porcine ovarian follicles, which were obtained on Days 5, 10, 15 and 18 of the oestrous cycle and at 24 h after the onset of oestrus. Concentrations of OMI decreased with increasing follicle diameter (P less than 0.05), independent of the stage of the oestrous cycle. Concentrations of inhibin showed a tendency to decrease with increasing follicle diameter on Days 10, 15 and 18, but not on Day 5 of the cycle. Concentrations of OMI and inhibin in the largest follicles were low before the onset of oestrus, and were essentially unaltered 24 h later. A positive correlation was found between OMI and inhibin concentrations, whereas the correlation between inhibin concentration and log (progesterone concentrations) was negative.     

Effects of follicular fluids on the growth of porcine preantral follicle and oocyte

We examined the effect of supplementing the culture medium with follicular fluid (FF) on the growth of porcine preantral follicles and oocytes. Firstly, preantral follicles were retrieved from ovaries and then FF was collected from all antral follicles that were 2-7 mm in diameter (AFF), which included large follicles of 4-7 mm in diameter (LFF) and small follicles of 2-3 mm in diameter (SFF). When preantral follicles with a diameter of 250 mum were cultured in medium containing AFF, the growth of follicles and oocytes was greater than when follicles were cultured in medium containing fetal calf serum (FCS). When this growth-promoting effect in AFF was compared for LFF and SFF, the LFF were shown to be significantly more effective than SFF. This LFF effect was lost, however, when the concentration of LFF in the medium was decreased from 5% to 0.5% or when LFF were heat treated (60 degrees C for 30 min) or trypsin was added. In contrast, a decrease in SFF concentration from 5% to 0.5% and heat treatment of the SFF enhanced preantral follicle growth. Furthermore, proteins obtained from LFF that had molecular weights greater than 10 kDa (LFF > 10 kDa) had similar, but relatively reduced, growth-promoting properties. The remaining three LFF protein fractions (<10 kDa or <100 kDa or >100 kDa), however, did not have these growth-promoting properties. In conclusion, the supplementation of medium with LFF, rather than serum, enhanced preantral follicle and oocyte growth. Factors that enhanced follicle development in LFF and factors that suppressed follicle development in SFF were proteins and these LFF factors ranged in size from 10 kDa to over 100 kDa.     

Preovulatory follicular fluid during in vitro maturation decreases polyspermic fertilization of cumulus-intact porcine oocytes in vitro maturation of porcine oocytes

Porcine follicular fluid (pFF), as a supplement of maturation media, has been shown several times to improve the in vitro production (IVP) of porcine embryos. As a transudate of serum, pFF contains locally produced factors in addition to the ones derived from serum. The objective of this study was to determine the additional positive effects of these pFF specific factors on the nuclear and cytoplasmic maturation of porcine oocytes. Follicular fluid and autologous serum were collected from sows in the preovulatory phase of the estrous cycle. Subsequently, oocytes from prepubertal gilts were matured in NCSU23 supplemented with either 10% pFF or 10% autologous serum derived from the same sow. Oocytes were then fertilized and the putative zygotes were cultured for 7 days. Nuclear maturation and cumulus expansion were assessed after the maturation culture. For evaluation of cytoplasmic maturation, oocyte glutathione (GSH) content, fertilization parameters and embryonic development were evaluated. After in vitro maturation (IVM) of the oocytes, both cumulus expansion rate and oocyte GSH content were increased for oocytes matured in pFF (P<0.05). More monospermic penetration was found when cumulus-intact oocytes had been matured in 10% pFF but this effect was lost after fertilization of cumulus denuded oocytes indicating that the pFF was acting through the cumulus. We speculate that the increased cumulus expansion and increased glutathione content, which were prevalent after IVM in pFF, are responsible for the positive effects on fertilization and the pre-implantation development of the embryos.     

The effects of porcine follicular fluid inhibin on gonadotropin secretion in the rabbit

Porcine follicular fluid (pff), treated with charcoal to remove steroids, was used to determine whether inhibin is active in the laboratory rabbit. When pff (5 ml/4 kg body weight) was injected (ip) into does that had been castrated 2 weeks earlier, there was a significant decline in blood follicle-stimulating hormone (FSH) levels; the decline lasted for 8-12 h. Blood levels of luteinizing hormone (LH) were suppressed, but only briefly at 3 h after injection. In other experiments, intact does which had been injected with pff 9 h and 10 min before receiving a single, i.v. injection of luteinizing hormone-releasing hormone (LHRH) (10 micrograms/kg body weight) showed a sharp reduction in the concentration of LH in the blood samples collected 15, 30 and 60 min after LHRH administration. Secretion of FSH responded poorly to LHRH stimulation, and pff had little suppressive action on blood levels. Having established that the pff preparation had inhibin activity, its action on the postovulatory surge of FSH secretion was next examined. This release of FSH, which occurs 6 to 36 h after ovulation, has been hypothesized to be required for the establishment of pregnancy by stimulating the growth of the ovarian follicles supplying the luteotropic estradiol. To test this hypothesis, pff was injected into rabbits every 8 h for the first 5 days of pregnancy and found to block the postovulatory FSH surge. The patterns of secretion of LH and progesterone in the same pff-injected animals were, however, not altered from normal pregnancy patterns by pff.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of extracellular potassium concentration on meiotic and cytoplasmic maturation in the porcine oocyte

Effects of extracellular potassium (K⁺) concentration in maturation media on the meiotic and cytoplasmic maturation of porcine oocytes were examined. Oocyte-cumulus cell complexes or cumulus cell denuded oocytes were cultured in Whitten's medium containing 0, 3, 6, 12 or 16 mM potassium. Absence of K⁺ in the media did not inhibit germinal vesicle breakdown (GVBD) in cumulus intact oocytes, but significantly decreased the frequency of meiotic maturation. In cumulus cell denuded oocytes, both GVBD and meiotic maturation were inhibited in K⁺-free medium. Millimole concentrations of K⁺ channel blockers, 4-aminopyridine or tetraethyl ammonium chloride inhibited GVBD and almost completely suppressed progression of meiotic maturation. The effect of varying the concentration of K⁺ on cytoplasmic maturation of pig oocytes was evaluated by the ability to form a male pronucleus after in vitro fertilisation. The percentage of sperm penetration or monospermic penetration was not different among treatments (P > 0.1). However, male pronuclear formation in oocytes in medium with 6 mM K⁺ was higher than in media with 12 and 16 mM K⁺. These results suggest that extracellular K⁺ is required for GVBD and meiotic maturation, and high concentrations (12 or 16 mM) of K⁺ in maturation media impair cytoplasmic maturation.     

Changes in histone acetylation during oocyte meiotic maturation in the diabetic mouse

Although there is considerable evidence that diabetes can adversely affect meiosis in mammalian oocytes, acetylation status of oocytes in a diabetic environment remains unclear. The objective was to determine acetylation or deacetylation patterns (based on immunostaining) of H3K9, H3K14, H4K5, H4K8, H4K12, and H4K16 sites at various stages during meiosis in murine oocytes from control and diabetic mice. According to quantitative real time polymerase chain reaction (qPCR), mean ± SEM relative expression of Gcn5 (1.70 ± 0.14 at metaphase [M]I and 1.27 ± 0.01 at MII, respectively), Ep300 (1.74 ± 0.04 at MI and 1.80 ± 0.001 at MII), and Pcaf (2.01 ± 0.03 at MI and 1.41 ± 0.18 at MII) mRNA in oocytes from diabetic mice were higher than those from controls (P < 0.05), whereas there was no difference (P > 0.05) during the germinal vesicle (GV) stage between the two groups (1.23 ± 0.04 for Gcn5, 0.82 ± 0.06 for Ep300, and 0.80 ± 0.07 for Pcaf). Conversely, relative mRNA expression concentrations of Hdac1, Hdac2, Hdac3, Sirt1 and Sirt2 during the germinal vesicle stage were lower in oocytes of diabetic mice (0.24 ± 0.03 for Hdac1, 0.11 ± 0.001 for Hdac2, 0.31 ± 0.03 for Hdac3, 0.28 ± 0.02 for Sirt1, and 0.55 ± 0.02 for Sirt2; P < 0.05). Similarly, the expression concentrations of these genes at the MI stage were lower in oocytes from diabetic mice (0.79 ± 0.12 for Hdac1, 0.72 ± 0.001 for Hdac2, 0.02 ± 0.001 for Sirt1, and 0.84 ± 0.08 for Sirt2; P < 0.05). Their expression concentrations at the MII stage were also lower in oocytes from diabetic mice (0.46 ± 0.03 for Hdac1, 0.93 ± 0.01 for Hdac2, 0.56 ± 0.01 for Hdac3, 0.01 ± 0.002 for Sirt1, and 0.84 ± 0.04 for Sirt2; P < 0.05). At the MI stage, however, there was no difference in the expression of Hdac3 between the two groups of oocytes (0.96 ± 0.03; P > 0.05). Taken together, diabetes altered the intracellular histone modification system, which may have contributed to changes in histone acetylation, and may be involved in the compromised maturation rate of oocytes in diabetic humans.     

SIRT4 is essential for metabolic control and meiotic structure during mouse oocyte maturation

SIRT4 modulates energy homeostasis in multiple cell types and tissues. However, its role in meiotic oocytes remains unknown. Here, we report that mouse oocytes overexpressing SIRT4 are unable to completely progress through meiosis, showing the inadequate mitochondrial redistribution, lowered ATP content, elevated reactive oxygen species (ROS) level, with the severely disrupted spindle/chromosome organization. Moreover, we find that phosphorylation of Ser293‐PDHE1α mediates the effects of SIRT4 overexpression on metabolic activity and meiotic events in oocytes by performing functional rescue experiments. By chance, we discover the SIRT4 upregulation in oocytes from aged mice; and importantly, the maternal age‐associated deficient phenotypes in oocytes can be partly rescued through the knockdown of SIRT4. These findings reveal the critical role for SIRT4 in the control of energy metabolism and meiotic apparatus during oocyte maturation and indicate that SIRT4 is an essential factor determining oocyte quality.     

Requirements of Src family kinase during meiotic maturation in mouse oocyte

The Src family kinase (SFK) is important in normal cell cycle control. However, its role in meiotic maturation in mammalian has not been examined. We used confocal microscope immunofluorescence to examine the in vitro dynamics of the subcellular distribution of SFK during the mouse oocyte meiotic maturation and further evaluated the functions of SFK via biochemical analysis using a specific SFK pharmacological inhibitor, PP(2). Our results showed that nonphospho-SFK was absent in oocyte upon its release from follicle. Nonphospho-SFK appeared in cytoplasm 0.5 hr after the release of oocyte and translocated to germinal vesicle (GV) before germinal vesicle breakdown (GVBD). After GVBD, nonphospho-SFK colocated with condensed chromosomes. In occyte at metaphase I (MI) and telophase I, nonphospho-SFK accumulated in the cortex and the cleavage furrow respectively besides its existence in cytoplasm in both stages. In oocyte at metaphase II (MII), nonphospho-SFK concentrated at the aligned chromosomes. In contrast, phospho-SFK was absent in oocyte until 1 hr after its release from the follicle. Phospho-SFK accumulated in the GV, the cortex, and cytoplasm immediately prior to GVBD. After GVBD, phospho-SFK evenly distributed in oocyte. In oocyte at MII, phospho-SFK localized throughout the cytoplasm and under the egg member. When the SFK activity was inhibited, the oocyte failed to initiate GVBD, could not go into MII, and could not extrude the first polar body. Our results demonstrated that SFK is required for meiotic maturation in mouse oocyte.     

Characterization of polo-like kinase 1 during meiotic maturation of the mouse oocyte

We have characterized plk1 in mouse oocytes during meiotic maturation and after parthenogenetic activation until entry into the first mitotic division. Plk1 protein expression remains unchanged during maturation. However, two different isoforms can be identified by SDS-PAGE. A fast migrating form, present in the germinal vesicle, seems characteristic of interphase. A slower form appears as early as 30 min before germinal vesicle breakdown (GVBD), is maximal at GVBD, and is maintained throughout meiotic maturation. This form gradually disappears after exit from meiosis. The slow form corresponds to a phosphorylation since it disappears after alkaline phosphatase treatment. Plk1 activation, therefore, takes place before GVBD and MAPK activation since plk1 kinase activity correlates with its slow migrating phosphorylated form. However, plk1 phosphorylation is inhibited after treatment with two specific p34(cdc2) inhibitors, roscovitine and butyrolactone, suggesting plk1 involvement in the MPF autoamplification loop. During meiosis plk1 undergoes a cellular redistribution consistent with its putative targets. At the germinal vesicle stage, plk1 is found diffusely distributed in the cytoplasm and enriched in the nucleus and during prometaphase is localized to the spindle poles. At anaphase it relocates to the equatorial plate and is restricted to the postmitotic bridge at telophase. After parthenogenetic activation, plk1 becomes dephosphorylated and its activity drops progressively. Upon entry into the first mitotic M-phase at nuclear envelope breakdown plk1 is phosphorylated and there is an increase in its kinase activity. At the two-cell stage, the fast migrating form with weak kinase activity is present. In this work we show that plk1 is present in mouse oocytes during meiotic maturation and the first mitotic division. The variation of plk1 activity and subcellular localization during this period suggest its implication in the organization and progression of M-phase.     

Localization and function of the Ska complex during mouse oocyte meiotic maturation

The Ska (spindle and kinetochore-associated) complex is composed of three proteins: Ska1, Ska2 and Ska3. It is required for stabilizing kinetochore-microtubule (KT-MT) interactions and silencing spindle checkpoint during mitosis. However, its roles in meiosis remain unclear. The present study was designed to investigate the localization and function of the Ska complex during mouse oocyte meiotic maturation. Our results showed that the localization and function of Ska complex in mouse oocyte meiosis differ in part from those in mitosis. Injection of low dose exogenous Myc-Ska mRNA showed that, instead of localizing to the kinetochores (KTs) and mediating KT-MT interactions from pro-metaphase to mid-anaphase stages as in mitosis, the members of the Ska complex were only localized on spindle microtubules from the Pro-MI to MII stages in mouse oocyte meiosis. Time-lapse live imaging analysis showed that knockdown of any member of the Ska complex by Morpholino injection into mouse oocytes resulted in spindle movement defects and enlarged polar bodies. Depletion of the whole Ska complex disrupted the stability of the anaphase spindle and influenced the extrusion of the first polar body. Taken together, these results show that the Ska complex plays an important role in meiotic spindle migration and anaphase spindle stability during mouse oocyte maturation.