Chromatin and cytoplasmic characteristics of equine oocytes recovered by transvaginal ultrasound-guided follicle aspiration are influenced by the developmental stage of their follicle of origin.

Dynamic follicular changes occur during the equine estrus cycle, but little is known about their impact on the properties of recovered oocytes. The aim of this study was to characterize the cytoplasmic and chromatin status of equine oocytes in relation to the time of recovery during the follicle wave. Transvaginal ultrasound-guided follicle aspiration was performed two times in relation to the follicle wave: estrus-subordinate, from the subordinate follicles of mares in estrus, 24 hours after human chorionic gonadotropin stimulation of a dominant preovulatory follicle, and new-wave, from the follicles of the subsequent induced follicular wave, at the time of dominant follicle divergence (largest follicle 23 mm diameter). A total of 1011 follicles were aspirated. The oocyte recovery rate in the new-wave group was significantly lower than that for the estrus-subordinate group (12% vs. 26%, respectively); this was associated with a significantly higher proportion of oocytes with compact cumuli (44% vs. 27%, respectively). Estradiol concentrations were markedly higher in follicular fluid from new-wave follicles (885.6 ± 123.2 ng/mL vs. 54.3 ± 18.9 ng/mL, for estrus-subordinate; P < 0.001), indicating greater viability. Aspiration group did not affect glucose-6-phosphate dehydrogenase activity in recovered oocytes. Fibrillar (more juvenile) chromatin was more prevalent in new-wave oocytes, whereas estrus-subordinate oocytes showed more condensed chromatin or resumption of meiosis (P < 0.05). Mitochondrial activity was higher in oocytes with expanded cumuli in the new-wave group, but not in the estrus-subordinate group. In conclusion, our results clearly showed that the time of aspiration in relation to the follicle wave is associated with significant differences in follicle status and oocyte characteristics: new-wave oocytes were from a more viable follicle population and had more juvenile chromatin and cytoplasmic characteristics, whereas estrus-subordinate oocytes were from a more atretic follicle population and exhibited signs of atresia-related acquisition of meiotic and cytoplasmic competence. These findings will help in effective scheduling of oocyte recovery for equine-assisted reproduction techniques.

Sperm migration in pigs after deep intrauterine and intraperitoneal insemination.

Deep intrauterine insemination in pigs allows sperm deposition only into one uterine horn, but bilateral fertilization of oocytes occurs. How the sperm reach the contralateral oviduct remains disputable. The aim of this experiment was to study possible transperitoneal and/or transuterine sperm migration ways. Follicle growth and ovulation were induced in 24 peripubertal gilts with eCG and hCG 72 h after eCG. Endoscopic intrauterine insemination (IUI) was performed 32 h after hCG with 20 ml of extended semen (60 × 10(6) spermatozoa) as follows: Group CONTROL (n=8) received IUI into the right horn, and the left horn served as non-treated control; Group LIGATURE (n=8) received IUI into the right horn, and the left horn was closed by endoscopic double ligature close to the bifurcation; Group INTRAPERITONEAL (IPI; n=8) received IUI into the right uterine horn, the left horn was closed by double ligature and semen was deposited intraperitoneally at the surface of the left ovary. Genital tracts were removed 65-66 h after hCG, the oviducts were flushed and ova (n=299) were analyzed for fertilization and cleavage. Furthermore, the accessory spermatozoa count/oocyte was graded as 0, without spermatozoa, 1, <5 spermatozoa, 2, 5-50 spermatozoa, 3, 50-100 spermatozoa and 4, >100 spermatozoa. The results indicate that low dose IUI into one horn provides a lower grade of accessory spermatozoa in the contra-lateral side (1.6 vs. 2.8). No spermatozoa were found in ova flushed from oviducts of the ligated uterine horn, even after intraperitoneal insemination (P<0.05), and no fertilization occurred, respectively. Our results clearly indicate that after low dose IUI into one uterine horn, spermatozoa reach the contralateral oviduct via transuterine migration.

Occurrence of polyovular follicles in mouse lines selected for high fecundity.

The aim of this study was to evaluate the prevalence of follicles with more than one oocyte (polyovular follicles, POFs) in mouse lines selected for high fecundity. The ovaries of 18 mice, 6 each from 3 different lines, were examined to evaluate the number of POFs and the follicular histology. Polyovular follicles were observed in the two high fecundity breeds, FL1 and FL2, as well as in the unselected control line, DUKsi. The highest number of POFs per ovary (27.0 +/- 7.2) was found in the FL1 line. The FL2 and DUKsi lines had 1.9 +/- 0.7 and 0.6 +/- 0.3 polyovular follicles per ovary, respectively. Most of the POFs contained 2 oocytes (>80%), but occasionally follicles containing up to 7 oocytes were observed. Follicles with more than 2 oocytes were observed in the FL1 line only.

Meiotic progression, mitochondrial features and fertilisation characteristics of porcine oocytes with different G6PDH activities.

The aim of the present study was to investigate the developmental competence, mitochondrial characteristics and chromatin status of immature follicular porcine oocytes selected for their glucose-6-phosphate dehydrogenase (G6PDH) activity by brilliant cresyl blue (BCB) staining. In Experiment 1, the oocyte parameters were determined in parallel right after BCB staining (T(0)), after 22 h of in vitro maturation (IVM) (T(22)) and after 44 h of IVM (T(44)) (n = 496). BCB-stained oocytes (BCB+) at T(0) were characterised by fibrillated chromatin filaments in their germinal vesicles (GV) and diakinesis stages whereas unstained (BCB-) oocytes at T(0) contained in their GV mainly condensed stages of chromatin (P < 0.05). After 22 h of IVM BCB+ oocytes showed a prominent chromatin configuration of metaphase I and after 44 h the majority developed a M II nuclear configuration in contrast to the BCB- group (P < 0.0001). Differences were also observed between the two oocyte populations in their mitochondrial activity (P < 0.05). At the beginning of IVM BCB+ oocytes were characterised by high mitochondrial activity in their cytoplasm. The BCB+ oocytes showed clear visible homogenous distributions of mitochondria (P < 0.005) and contained more aggregated clusters of mitochondria in contrast to BCB- oocytes (P < 0.005). In Experiment 2, 318 oocytes were tested for their G6PDH activity and introduced to IVM and IVF. Only oocytes from the BCB+ group, which were matured after 44 h up to the stage of M II (81.6%) were fertilised (17.4%), penetrated (46%) or activated (15.6%) after IVF. These results indicate a relationship between the G6PDH activity of porcine oocytes before IVM and their subsequent nuclear development, mitochondrial activity and aggregation.

Regulation of cap-dependent translation initiation in the early stage porcine parthenotes.

The binding of mRNAs to ribosomes is mediated by the protein complex eIF4F in conjunction with eIF4B (eukaryotic initiation factor 4F and 4B). EIF4F is a three subunit complex consisting of eIF4A (RNA helicase), eIF4E (mRNA cap binding protein), and eIF4G (bridging protein). The crucial role is played by eIF4E, which directly binds the 5'-cap structure of the mRNA and facilitates the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. EIF4E binding to mRNA and to other initiation factors is regulated on several levels, including its phosphorylation on Ser-209, and association with its regulatory protein 4E-binding protein (4E-BP1). In this study we document that both the translation initiation factor eIF4E and its regulator 4E-BP1 become dephosphorylated in the early stage porcine zygotes already 8 hr post-activation. Similarly, the activities of ERK1/2 MAP and Mnk1 kinases, which are both involved in eIF4E phosphorylation, gradually decrease during this period with the timing similar to that of eIF4E dephosphorylation. The formation of an active eIF4F complex is also diminished after 9-15 hr post-activation, although substantial amounts of this complex have been detected also 24 hr post-activation (2-cell stage). The overall protein synthesis in the parthenotes decreases gradually from 12 hr post-activation reaching a minimum after 48 hr (4-cell stage). Although the translation is gradually decreasing during early preimplantation development, the eIF4F complex, which is temporarily formed, might be a premise for the translation of a small subset of mRNAs at this period of development.

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.

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

BACKGROUND: Oocyte 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. RESULTS: Comparative 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. CONCLUSION: This 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.

Effect of recombinant bovine somatotropin (rbST) on cytoplasmic maturation of bovine oocytes and their developmental competence in vitro.

The objectives of this study were to evaluate the effects of recombinant bovine somatotropin (rbST) on the nuclear and cytoplasmic maturation of bovine oocytes and their further developmental competence to blastocysts in vitro. We analyzed the mitochondrial activity and concentration of intracellular stored calcium ([Ca(2+)](is)) in matured oocytes and the morphology and chromatin status of produced embryos after in vitro fertilization. Cumulus-oocyte complexes were incubated in TCM 199 containing 10% fetal calf serum (control medium 1: CM 1) or 10% estrus cow serum (control medium 2: CM 2). The culture medium of the treatment groups was modified by supplementation of the control medium with 10 ng/ml rbST (CM 1A and CM 2A), 10(6)/ml granulosa cells (CM 1B and CM 2B), or 10 ng/ml rbST plus 10(6)/ml granulosa cells (CM 1C and CM 2C). No differences were observed in the percentages of oocytes reaching metaphase II between the groups. However, the proportion of blastocysts was highest in treatment groups CM 1C and CM 2C (P<0.05). The type of serum did not alter the positive effect of rbST on the developmental competence of embryos. The fluorescence intensity of metabolically active mitochondria measured by intensity per oocyte (Em 570) after MitoTracker CMTM Ros Orange labeling was significantly increased in oocytes matured in the presence of 10 ng/ml rbST and granulosa cells (309.21 vs. 119.97 microA; P<0.01). In parallel, the concentration of [Ca(2+)](is) in oocytes, determined using fluorophore chlortetracycline, was significantly decreased (0.85 +/- 0.02 vs. 0.97 +/- 0.03 AU; P<0.05). Based on these results, we concluded that rbST, in interaction with granulosa cells stimulates the oxidative activity of ooplasmic mitochondria and decreases the content of [Ca(2+)](is) in oocytes. These facts support the hypothesis that somatotropin influences the developmental competence of bovine oocytes during maturation in vitro, and this effect can be modulated by granulosa cells.

Experimental evidence for the influence of cumulus-oocyte-complexes on sperm release from the porcine oviductal sperm reservoir.

In the pig, a temporal relationship is suggested between sperm release from the sperm reservoir (SR) and ovulation, but the mechanism(s) is still under discussion. In two experiments, the influence of transferred ova on the release of SR-spermatozoa at ovulation and the effect of supplementation with non-sulfated glycosaminoglycan hyaluronan (HA) on embryo development and the number of accessory spermatozoa, respectively, were examined. PMSG/hCG primed ovectomized gilts that had previously received endoscopic low-dose insemination into the cranial uterine horn were used as an experimental model. After salpingectomy, tubal segments (ampulla, cranial, and caudal isthmus) were flushed and sperm numbers or respective accessory spermatozoa were counted. In Experiment 1, the distribution of the sperm population was altered in the presence of cumulus-oocyte-complexes (COCs). A higher proportion of spermatozoa was found after transfer of COCs into one oviduct in the ampulla and cranial isthmus segments compared with the controls (17.5 vs. 4.9%, p<0.05). In Experiment 2, the quality of the transferred ova and treatment influenced the presence of accessory spermatozoa. Transfer of COCs together with HA increased (p<0.05) the number of accessory spermatozoa compared with the other treatment groups and was similar to those in the "undisturbed" controls. No modifications were obtained regarding mean blastomere numbers (2.6 +/- 0.2 to 3.1 +/- 0.2). In summary, this study was demonstrated that cumulus-oocyte-complexes may be involved in triggering sperm release from the pig oviductal SR and that HA might be related to sperm release.

Developmental competence of HMC(TM) derived bovine cloned embryos obtained from somatic cell nuclear transfer of adult fibroblasts and granulosa cells.

To enable us to handle a large number of oocytes at a given time and to have an increased throughput of cloned embryos, we attempted the Handmade cloning (HMC) technique, a zona-free method of bovine somatic cell nuclear transfer. Our objective was to study the developmental competence of the HMC derived embryos obtained using different types of somatic cells. A total of 6,874 cumulus-oocyte-complexes were used with either 7th or 11th passage fibroblasts (1st and 2nd groups, respectively), which were prepared from male animals, or granulosa cells (3rd group) as nuclei donors. The average cleavage rate was 65%, accompanied by a blastocyst rate of just 2% for the cleaved products and 5% for the >8-cell embryos, and there was no significant difference between the three groups. Out of 27 blastocysts recovered, 22 blastocysts were transferred to 22 recipients, resulting in two pregnancies. One pregnancy was lost after the fourth week while the other progressed to full term with the birth of a male calf. This first successful cloning of a male calf with the HMC technique in Europe indicates the successful adoption and establishment of this technique in our laboratory, and that this technique can be successful in producing viable embryos.

Comparison of follicular and oocyte development and reproductive hormone secretion during the ovulatory period in Hungarian native breed, Mangalica, and Landrace gilts.

Only a very small amount of physiological data is available about the low fertility (mean litter size is 5.7+/-0.8) of Hungarian native breed, Mangalica (M), sows. The aim of the present paper is to reveal the differences in preovulatory follicle development and intrafollicular oocyte maturation between M and Landrace (L) gilts, with special reference to the peri- and postovulatory secretion and peripheral concentrations of estradiol-17beta (E2), progesterone (P4), and luteinizing hormone (LH). The number of preovulatory follicles was 6.8+/-1.4 and 19.6+/-6.6 in M and L gilts, respectively. A lower degree of cumulus expansion and a lower percentage of mature oocytes (TI/M II) was noted in M. Higher LH and E2 peak levels, a longer E2 to LH peak interval, and lower embryo survival was confirmed. Interestingly, despite the lower number of corpora lutea, a higher peripheral blood level of P4 was shown in M than in L gilts. Both diminished follicular development and protracted oocyte maturation may be involved in low fecundity in M, and the present findings may explain these reproductive phenomena.

Ovarian activity and oocyte development during follicular development in pigs at different reproductive phases estimated by the repeated endoscopic method.

The aim of the present study was to assess follicular and oocyte development in the same gilts during three phases of their reproductive life [prepuberal gilts (PP; 6.0 months of age), puberal gilts (P; 9.5 months of age) and primiparous sows (S)]. Follicular development was stimulated by the injection of 1,000 IU of equine chorionic gonadotropin (eCG) followed by 500 IU of human chorionic gonadotropin (hCG) 72 h later. Cumulus-oocyte-complexes (COCs) were recovered by endoscopic ovum pick up/aspiration from preovulatory follicles of the left ovary, and the follicular fluid (FF) from the right ovary was collected 34 h after the hCG treatment by endoscopy. Altogether, 19 pigs were used in the PP and P trials and 12 in the S trial. From the left ovaries, 168, 190 and 82 follicles were aspirated and 106, 125 and 42 COCs, respectively, were recovered (recovery rate 61 +/- 27, 63 +/- 21 and 53 +/- 22%, respectively). The mean number of follicles was greater in the P phase than in the PP phase (19.7 +/- 6.8 vs. 15.7 +/- 6.8; p=0.06) and S phases (14.2 +/- 4.0; p<0.05). More uniform oocytes with an expanded cumulus were aspirated in the P and PP phases than in the S phase (90 and 78 vs. 46%; p<0.05). Furthermore, the meiotic configuration in oocytes (T I/M II stage) differed between the three phases (56 and 62 vs. 0%; p<0.05). Progesterone (P4) levels in FF decreased from 590.0 +/- 333.6 (PP) to 249.1 +/- 72.6 (P) and 161.4 +/- 75.2 ng/ml (S) (p<0.05). Estradiol-17beta (E2) levels differed between PP and P gilts and S sows (9.3 +/- 2.9, 21.9 +/- 10.6 and 94.0 +/- 15.9 pg/ml, respectively; p<0.05), and the P4/E2 ratio was 72, 15 and 5, respectively. These results indicate differences in follicular and oocyte development between the reproductive phases investigated. Puberal gilts should preferably be used in IVF and breeding programs. The lower reproductive potential of primiparous sows must be taken into consideration in breeding. Any prediction of lifetime performance based on individual ovarian reactions of prepuberal gilts is unreliable.