W. Kanitz

Molecular and subcellular characterisation of oocytes screened for their developmental competence based on glucose-6-phosphate dehydrogenase activity

Oocyte selection based on glucose-6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate between competent and incompetent bovine oocytes. However, the intrinsic molecular and subcellular characteristics of these oocytes have not yet been investigated. Here, we aim to identify molecular and functional markers associated with oocyte developmental potential when selected based on G6PDH activity. Immature compact cumulus-oocyte complexes were stained with brilliant cresyl blue (BCB) for 90 min. Based on their colouration, oocytes were divided into BCB(-) (colourless cytoplasm, high G6PDH activity) and BCB(+) (coloured cytoplasm, low G6PDH activity). The chromatin configuration of the nucleus and the mitochondrial activity of oocytes were determined by fluorescence labelling and photometric measurement. The abundance and phosphorylation pattern of protein kinases Akt and MAP were estimated by Western blot analysis. A bovine cDNA microarray was used to analyse the gene expression profiles of BCB(+) and BCB(-) oocytes. Consequently, marked differences were found in blastocyst rate at day 8 between BCB(+) (33.1+/-3.1%) and BCB(-) (12.1+/-1.5%) oocytes. Moreover, BCB(+) oocytes were found to show higher phosphorylation levels of Akt and MAP kinases and are enriched with genes regulating transcription (SMARCA5), cell cycle (nuclear autoantigenic sperm protein, NASP) and protein biosynthesis (RPS274A and mRNA for elongation factor 1alpha, EF1A). BCB(-) oocytes, which revealed higher mitochondrial activity and still nucleoli in their germinal vesicles, were enriched with genes involved in ATP synthesis (ATP5A1), mitochondrial electron transport (FL405), calcium ion binding (S100A10) and growth factor activity (bone morphogenetic protein 15, BMP15). This study has evidenced molecular and subcellular organisational differences of oocytes with different G6PDH activity.

Effect of sperm cryopreservation and treatment with calcium ionophore or heparin on in vitro fertilization of horse oocytes.

Little information is available on methods of sperm capacitation for IVF in the horse. In this study, we summarized results of several independent trials that compared acrosome reaction, hyperactivation and chromatin integrity of fresh or cryopreserved stallion spermatozoa after treatment with heparin or with calcium ionophore. We also examined the influence of spermatozoa storage (fresh vs. cryopreserved), capacitation treatment, oocyte maturation time and cumulus morphology on the penetration rate and fertilization rate. We recovered cumulus-oocyte-complexes (COCs) from ovaries by ultrasound guided follicle aspiration or by scraping of follicles from ovaries obtained at a slaughterhouse. Upon recovery, we evaluated the cumulus morphology, and the COCs were matured in vitro for 18 to 24 or 26 to 40 h. Fresh semen and cryopreserved semen were treated either with heparin (200 microg/mL) or calcium ionophore (7.14 microM). Overall, 28.4% (99/349) of the oocytes were penetrated, and 12.9% (45/349) were fertilized. Fresh spermatozoa treated with calcium ionophore showed a higher penetration rate than cryopreserved spermatozoa (36.0 vs. 0%). Fresh and heparin-treated spermatozoa showed a penetration rate of 29.1%, and the same treatment for cryopreserved spermatozoa showed a penetration rate of 33.7%; none of these differences was significant (P>0.05). Fertilization rates after the calcium and heparin treatment followed the same trend and also showed no significant differences. Prolonged maturation period resulted in higher penetration (P<0.05) and fertilization rates in compact (26 to 40 h: 37.7 and 13.1% vs. 18 to 24 h: 13.1 and 2.8%) and in tendency in expanded COCs (26 to 40 h: 40.0 and 30.3% vs. 18 to 24 h: 29.4 and 13.5%). In oocytes with only a few cumulus cells, the rates tended to be higher after the shorter incubation (18 to 24 h: 33.5 and 18.8% vs. 26 to 40 h: 17.2 and 6.5%). We observed hyperactivation more frequently in fresh than in cryopreserved semen after different treatments (43.2, 39.1 and 35.4% for heparin, calcium ionophore and control vs. 15.7, 10.8 and 5.7%, respectively). We observed significant changes in the acrosome reaction of fresh spermatozoa after heparin treatment (62.6 vs. 48.2%, P<0.05), as well as in cryopreserved spermatozoa after calcium ionophore treatment (31.7 vs. 17.6%, P<0.05). The chromatin integrity was significantly reduced after heparin treatment of fresh spermatozoa, in comparison to control and calcium ionophore (81.0 vs. 87.3 and 86.6, P<0.02). We also observed a similar reduction of chromatin quality after heparin treatment in cryopreserved spermatozoa, but the difference was significant only between heparin and calcium ionophore treatment [77.4 vs. 86.4 (P<0.02) and 84.9]. The results in the this retrospective study show that capacitating fresh spermatozoa with calcium ionophore, or using heparin in cryopreserved spermatozoa, results in higher penetration and fertilization rates of in vitro matured horse oocytes. A prolonged maturation time of 26 to 40 h is necessary for compact cumulus oocyte complexes to achieve the fertilization capacity. Further investigation is needed to show the developmental capacity of these fertilized oocytes.

Influence of organochlorine pesticides on maturation and postfertilization development of bovine oocytes in vitro.

The aim of this study was to perform a dose-response test to determine whether bovine oocytes exposed to dichlorodiphenyltrichloroethane (DDT), hexachlorocyclohexane (gammaHCH), or methoxychlor (MXC) in vitro would exhibit changes in maturation rates, cleavage rates at Day 2, or blastocyst rates at Day 7 to 8 after fertilization in vitro (IVF). All three pesticides affected maturation and degeneration rates in a dose-dependent manner, but to different extents. Higher concentrations of pesticides were associated with higher rates of chromatin degeneration. Because the maturation of bovine oocytes was depressed in a dose-dependent manner, the fertilizability and further embryonic development of in vitro matured oocytes was studied at the lowest previously tested concentration (7.25 microg/mL) only. No significant difference in fertilization rates was seen between unexposed control and treated groups. The cleavage rates did not differ among groups 48 h after IVF. The number of morulae and blastocysts on Day 7 to 8 after IVF, which is commonly used as a parameter for normal development, was significantly different between control and DDT- and gammaHCH-treated groups, but not between the control and MXC groups. The pesticides did not differ significantly among themselves. These results show that the tested pesticides decrease the rate of normal oocyte maturation in vitro in a dose-dependent manner. The effect of the lowest concentration of pesticides is seen only after Day 7 of embryo development.

Alterations in transcript abundance of bovine oocytes recovered at growth and dominance phases of the first follicular wave

BackgroundOocyte developmental competence is highly affected by the phase of ovarian follicular wave. Previous studies have shown that oocytes from subordinate follicles recovered at growth phase (day 3 after estrus) are developmentally more competent than those recovered at dominance phase (day 7 after estrus). However, the molecular mechanisms associated with these differences are not well elucidated. Therefore, the objective of this study was to investigate transcript abundance of bovine oocytes retrieved from small follicles at growth and dominance phases of the first follicular wave and to identify candidate genes related to oocyte developmental competence using cDNA microarray.ResultsComparative gene expression analysis of oocytes from growth and dominance phases and subsequent data analysis using Significant Analysis of Microarray (SAM) revealed a total of 51 differentially regulated genes, including 36 with known function, 6 with unknown function and 9 novel transcripts. Real-time PCR has validated 10 transcripts revealed by microarray analysis and quantified 5 genes in cumulus cells derived from oocytes of both phases. The expression profile of 8 (80%) transcripts (ANAXA2, FL396, S100A10, RPL24, PP, PTTG1, MSX1 and BMP15) was in agreement with microarray data. Transcript abundance of five candidate genes in relation to oocyte developmental competence was validated using Brilliant Cresyl Blue (BCB) staining as an independent model. Furthermore, localization of mRNA and protein product of the candidate gene MSX1 in sections of ovarian follicles at days 0, 1, 3 and 7 of estrous cycle showed a clear fluorescent signal in both oocytes and cumulus cells with higher intensity in the former. Moreover, the protein product was detected in bovine oocytes and early cleavage embryos after fertilization with higher intensity around the nucleus.ConclusionThis study has identified distinct sets of differentially regulated transcripts between bovine oocytes recovered from small follicles at growth and dominance phases of the first follicular wave. The validation with independent model supports our notion that many of the transcripts identified here may represent candidate genes associated with oocyte developmental competence. Further specific functional analysis will provide insights into the exact role of these transcripts in oocyte competence and early embryonic development.

Relationship between different stages of the corpus luteum and the expression of the peroxisome proliferator-activated receptor gamma protein in bovine large lutein cells.

Lutein cells produce progestins that support pregnancy. Steroidogenesis requires coordination of the anabolic and catabolic pathways of lipid metabolism. Peroxisome proliferator-activated receptors (PPAR) are transcription factors that are central in the regulation of lipid metabolism. Hence, they may play a role in regulation of the development and regression of the corpus luteum. The present study investigated the expression of PPAR-gamma, n during different stages of the corpus luteum. Lutein cells were isolated mechanically from non-pregnant and pregnant heifers on days 5, 12 and 20 of the oestrous cycle (n = 3 for each day). PPAR-gamma in single cells was analysed by flow cytometry. PPAR-gamma 1 and PPAR-gamma 2 isoforms were distinguished by immunoblotting. The cell cycle of the lutein cells was measured by the flow cytometric quantification of DNA in single cells, using propidium iodide staining after ethanol fixation and RNAse treatment, and by the detection of the proliferating cell nuclear antigen (PCNA). The response of the cells to PPAR-gamma agonist 15-deoxy-delta 12,14 prostaglandin J2 (15dPGJ2, 200 and 490 nmol l-1) with and without changing the cell cycle by the anti-apoptogenic drug aurintricarboxylic acid (ATA, 10 mumol l-1) was used as an in vitro model to study the relationship between the cell cycle and PPAR-gamma. The concentration of PPAR-gamma per cell from non-pregnant heifers was significantly higher on day 5 (3.40 +/- 0.30 fmol) compared with that on day 12 (1.34 +/- 0.18 fmol, P < 0.05) and day 20 (0.55 +/- 0.2 fmol, P < 0.05). In pregnant heifers, the concentration of PPAR-gamma was significantly (P < 0.01) higher than in non-pregnant heifers. A decrease in the PPAR-gamma 1 isoform relative to PPAR-gamma 2 was observed in cells on day 12 of the oestrous cycle compared with day 5. The cell cycle (S phase portion in cells on days 5, 12 and 20: 16 +/- 4%, 6 +/- 4% and 4 +/- 3%, respectively) and the portion of cells with PCNA correlated with the amount of PPAR-gamma in non-pregnant heifers. ATA promoted the S phase in cells of non-pregnant heifers (day 12) and the endogenous agonist of PPAR-gamma, 15dPGJ2, inhibited the response to ATA in a dose-dependent manner, indicating that PPAR-gamma plays a role in the arrest of the cell cycle in lutein cells to maintain their differentiated state.

MORPHOLOGY OF PORCINE CUMULUS-OOCYTE-COMPLEXES DEPENDS ON THE STAGE OF PREOVULATORY MATURATION

The aim of this investigation was to determine the relationship between the morphology of the cumulus-oocyte-complexes (COCs) and the meiotic configuration of oocytes as an LH peak mimicked by hCG. Estrus was synchronized in a total of 29 crossbred Landrace gilts by feeding Regumate for 15 d and administering 1000 IU PMSG. The LH peak was simulated by treatment with 500 IU hCG at 80 h after PMSG. Endoscopic oocyte recovery was carried out 2 h before and 10, 22 and 34 h after hCG. Only macroscopically healthy follicles with a diameter of more than 5 mm were punctured. Altogether, 410 follicles from 57 ovaries were punctured and 251 COCs were aspirated. Oocyte recovery rate increased from 48.5% (P < 0.01) of the early, not yet preovulatory follicles (2 h before hCG) to 80.8% of late preovulatory follicles (34 h after hCG). Cumulus morphology in COCs recovered 2 h before and 10 h after hCG was heterogeneous, with most (72.9 to 57.4%; P < 0.01) showing a compact or slightly expanded cumulus. Starting at about 22 h after hCG, COC morphology changed dramatically (86.7% of COCs with expanded cumulus; P < 0.01), and 34 h after hCG, 98.3% of the COCs had only an expanded cumulus. The percentage of oocytes with a mature meiotic configuration increased (11.2; 7.1; 41.4 and 70.2%, respectively, n = 238 oocytes; P < 0.01) as the interval post hCG increased (-2, 10, 22, 34 h, respectively). Meiotic configuration was related to COC morphology: compact COCs--88.9% diplotene, expanded COCs--53.8% metaphase II (M-II), and denuded oocytes--69.2% degenerated chromatin. These results indicate that there is a relationship between oocyte recovery rate, COC morphology, and meiotic configuration and preovulatory follicle maturation after the application of hCG.

Regulation of the expression and bioactivation of the epidermal growth factor receptor system by estradiol in pig oviduct and endometrium

Growth factors, such as epidermal growth factor (EGF), have been suggested to mediate local effects of steroid hormones within female reproductive tissue. In the present study, the influence of estrogen on the expression and bioactivity of the EGF receptor (EGF-R) system was investigated in pigs. Oviducal and endometrial tissue from gilts was analysed either at two different time points after ovulation (Day 12 and Day 20), or from ovariectomized animals, with or without steroid-replacement treatment. Estrogen receptor protein concentrations were significantly down-regulated both in oviducal and endometrial tissue under estrogen-influence, in contrast to increased progesterone receptor concentrations. Transcript levels of EGF and transforming growth factor alpha remained unchanged in both the oviduct and endometrium during treatment. Oviducal EGF-R mRNA was found to be increased after estradiol treatment with concurrent increases in EGF-R protein. However, in endometrial tissue of estradiol-substituted ovariectomized pigs, the receptor transcript was significantly reduced, indicating a different regulation of EGF-R transcription within the endometrium. The bioactivity of the EGF-R, analysed by tyrosine kinase assays, was preserved throughout experiments in the porcine oviduct and endometrium without obvious changes caused by the steroids. In conclusion, estradiol may play a key role during the proliferation and differentiation of porcine oviducal tissue by activating the important paracrine or autocrine EGF system through its receptor. The cell-specific influence of progesterone during regulation of the EGF-R expression in the endometrium requires further investigation.

Analysis of mRNA associated factors during bovine oocyte maturation and early embryonic development

Regulation of gene expression at the translational level is particularly essential during developmental periods, when transcription is impaired. According to the closed‐loop model of translational initiation, we have analyzed components of the 5´‐mRNA cap‐binding complex eIF4F (eIF4E, eIF4G, eIF4A), the eIF4E repressor 4E‐BP1, and 3´‐mRNA poly‐(A) tail‐associated proteins (PABP1 and 3, PAIP1 and 2, CPEB1, Maskin) during in vitro maturation of bovine oocytes and early embryonic development up to the 16‐cell stage. Furthermore, we have elucidated the activity of distinct kinases which are potentially involved in their phosphorylation. Major phosphorylation of specific target sequences of PKA, PKB, PKC, CDKs, ATM/ATR, and MAPK were observed in M II stage oocytes. Furthermore, main changes in the abundance and/or phosphorylation of distinct mRNA‐binding factors occur at the transition from M II stage oocytes to 2‐cell embryos. In conclusion, the results indicate that, at the transition from oocyte to embryonic development, translational initiation is regulated by striking differences in the abundance and/or phosphorylation of 5´‐end and 3´‐end mRNA associated factors, mainly the poly‐(A) bindings proteins PABP1 and 3, their repressor PAIP2 and a Maskin‐like protein with distinct eIF4E‐binding properties which prevents eIF4E/cap binding and eIF4F formation in vitro. Nevertheless, from the M II stage to 16‐cell embryos a substantial amount of eIF4E and, to a lesser extent, of eIF4G was precipitated by 7m‐GTP‐Separose indicating eIF4F complex formation. Therefore, it is likely that in general the reduction in PABP1 and 3 abundance represses overall translation during early embryonic development. Mol. Reprod. Dev. 76: 1208–1219, 2009.

Comparison of repeated transvagtnal ovum pick up in heifers by ultrasonographic and endoscopic instruments

Endoscopically and ultrasonographically guided ovum pick up were studied in 15 heifers once per week. Althogether 60 sessions were carried out. The number of aspirated follicles per animal (13.0 vs 10.3) did not differ significantly between both methods. Similar recovery rates (39 % vs 44 %) were achieved after repeated sessions. As a consequence the number of recovered oocytes per animal (5.2 vs 4.7) were not significantly different. Inter-animal- and intra-animal-variations were more important for the results than the applied methods. In contrast to these findings the quality of recovered cumulus-oocyte-complexes (COC) was significantly influenced by the methods. The COC were divided into 4 categories a) oocytes with compact cumulus, b) oocytes with rest of compacted cumulus, c) oocytes with expanded cumulus and d) oocytes without cumulus. There was a higher denudation rate of COC when using the endoscopic aspiration (62.0 % vs 6.6 %) because of turbulent current in this aspiration system. Advantages and disadvantages of both methods are described and discussed. Generally, the ultra sonographic method is the less traumatic procedure for the vagina, fornix and for abdominal organs. The other method is less traumatic for the ovaries.

Influence of synthetic lamprey GnRH-III on gonadotropin release and steroid hormone levels in gilts.

Based on the supposition that lamprey GnRH-III (lGnRH-III) elicits FSH releasing activity in swine, synthetic lGnRH-III (peforelin, Maprelin® XP10) was used in puberal estrus synchronized gilts. The secretion of reproductive hormones FSH, LH, estradiol and progesterone was analyzed, and follicle growth and ovulation recorded. Altogether, 24 German Landrace gilts were treated after an 18-day long synchronization of the estrus cycle with Regumate® as follows: 48 h after the last Regumate® feeding they received im either 150 μg Maprelin® XP10 (lGnRH-III, group Maprelin, n = 6), 50 μg Gonavet Veyx® (GnRH-I agonist, group GnRH, n = 6), 850 IE Pregmagon® (eCG, group eCG, n = 6) or saline (group Control, n = 6). Additionally, in eight gilts the concentrations of FSH and LH were analyzed after treatment with 150 μg Maprelin® XP10 (n = 3), 50 μg Gonavet Veyx® (n = 3) or saline (n = 2) at mid-cycle (day 10 of the estrus cycle). Blood samples were collected via implanted jugular vein catheters. Ovarian features were judged endoscopically at the end of the Regumate® feeding and on days 5 and 6 after treatment. Maprelin® XP10 had no effect on FSH release in gilts; neither at the pre-ovulatory period or at mid-cycle. Furthermore, LH levels were unaffected. In contrast, GnRH-I agonist stimulates FSH release, however less compared to LH secretion. LH secretion was induced by GnRH-I both during the follicular phase and at mid-cycle. Equine CG did not stimulate the release of pituitary hormones FSH and LH due to its direct action on the ovary. Increased estradiol concentrations during days 2 to 5 after Regumate® in all treatment groups indicated pre-ovulatory follicle growth in gilts. Equine CG stimulated a higher (P < 0.01) number of ovulatory follicles compared to the other treatment groups. All together, 83 to 100 % of gilts ovulated by day 6 post treatment. In summary, results of our study on reproductive hormone secretion do not provide evidence that synthetic lGnRH-III (Maprelin® XP10) selectively releases FSH in estrus synchronized gilts.