Phosphorylation of histone H3 on Ser10 by auto-phosphorylated PAK1 is not essential for chromatin condensation and meiotic progression in porcine oocytes

Background

The p21-activated kinase 1 (PAK1) is essential for mitosis and plays an important role in the regulation of microtubule assembly during oocyte meiotic maturation in mice; however, little is known about its role in porcine oocytes.

Result

Total p21-activated kinase 1 (PAK1) and phosphorylated PAK1 at Thr423 (PAK1^Thr423) were consistently expressed in porcine oocytes from the germinal vesicle (GV) to the second metaphase (MII) stages, but phosphorylation of histone H3 at Ser10 (H3^Ser10) was only expressed after the GV stage. Immunofluorescence analysis revealed that PAK1^Thr423 and H3^Ser10 colocalized on chromosomes after the GV stage. Blocking of endogenous PAK1^Thr423 by injecting a specific antibody decreased the phosphorylation level of H3^Ser10; however, it had no impact on chromatin condensation, meiotic progression, cleavage rate of blastomeres or the rate of blastocyst formation.

Conclusion

Phosphorylation of PAK1^Thr423 is a spontaneous activation process and the activated PAK1^Thr423 can promote the phosphorylation of H3^Ser10; however, this pathway is not required for meiotic maturation of porcine oocytes or early embryonic development.     

Upregulation of P-glycoprotein activity in porcine oocytes and granulosa cells during in vitro maturation

Multidrug resistance P-glycoprotein (Pgp), coded by the multidrug resistance type I (MDR1/ABCB1) gene, is an energy-dependent efflux pump and functions in systemic detoxification processes. In the present study, the expression and development of Pgp were evaluated in the porcine oocyte during in vitro maturation to compare with the expression of Pgp in cultured granulosa cells. As revealed by Western blotting using anti-human Pgp antibody, a single band of Pgp with an apparent molecular size of 170 kDa was detected in the germinal vesicle stage oocytes. The surface of GV oocyte was positively labeled by immunostaining. In the second metaphase oocyte after culture in the maturation medium containing porcine follicular fluid and human chorionic gonadotropin, the level of Pgp was increased. The elevation of the oocyte Pgp level was associated with increased activity of rhodamine 6G efflux from the oocyte, and its efflux was suppressed by verapamil, an inhibitor of Pgp. Removal of porcine follicular fluid from the maturation medium resulted in little alteration of the oocyte Pgp level. Expression of Pgp was also elevated in cultured porcine granulosa cells during cell maturation when stimulated with follicle-stimulating hormone or luteinizing hormone for 24-48 h. Collectively, the present results indicate that the transporting activity of P-glycoprotein upregulates in porcine oocytes and granulosa cells during exposure to gonadotropins or prior to ovulation.     

Mitochondrial activity and morphology in developing porcine oocytes and pre-implantation non-cultured and cultured embryos

Mitochondria are important determinants of developmental competence for oocytes and embryos owing to their central role in cellular metabolism, yet mitochondrial activity and morphometry during early porcine development have not been quantified. In this study, we examined the membrane potential Δψ(m) and the surface density Sv(in,m) of the inner mitochondrial membrane in pig oocytes and pre-implantation embryos using fluorescent probes and confocal microscopy. Mitochondria and their cristae were also examined by transmission electron microscope. Δψ(m) was consistently low from immature oocytes up to morulae and increased significantly in the early blastocyst before decreasing at the expanded blastocyst stage. This stage-dependent pattern of Δψ(m) changes differs from that reported for other mammals. We also determined that Δψ(m) is lower in cultured when compared to non-cultured porcine early blastocysts. Sv(in,m) was higher in immature oocytes than mature oocytes and remained constant up to the 4- to 8-cell embryo stage. It increased significantly at morula and early blastocyst stages. No differences in Sv(in,m) were found between developmentally matched non-cultured and cultured embryos. These results indicate that the inner mitochondrial membrane potential and surface density change significantly during pre-implantation porcine development in relation to metabolic alterations of the embryo. It is possible that modification of Δψ(m) by manipulating culture conditions may improve the performance of embryos that develop in vitro.     

Porcine CPEB1 is involved in Cyclin B translation and meiotic resumption in porcine oocytes

Ovarian immature oocytes accumulate many dormant maternal mRNAs, which have short poly(A) tails. Cytoplasmic‐polyadenylation‐element binding protein (CPEB) has been reported to play key roles for the elongation of the tails and the translation of these mRNAs in Xenopus oocytes. However, the functions of CPEB in meiotic resumption have not yet been established in mammalian oocytes. The present study examined the roles of porcine CPEB in Cyclin B syntheses and meiotic resumption of porcine oocytes. Porcine CPEB1 (pCPEB1) cDNA was cloned from total RNA of immature oocytes by RT‐PCR. The overexpression of pCPEB1 by mRNA injection into immature oocytes increased Cyclin B expression and the rate of meiotic resumption. Conversely, the inhibition of endogenous CPEB by expression of a dominant‐negative mutant pCPEB1 (AA‐CPEB), which replaced the expected phosphorylation sites with alanines, had the effect of inhibiting Cyclin B synthesis, ribosomal S6 kinase phosphorylation (an indicator of Mos activity), and meiotic resumption. The inhibition of porcine Aurora A by an injection of antisense RNA enhanced the inhibitory effects of AA‐CPEB. These results suggest the involvement of mammalian CPEB1 in Cyclin B syntheses and meiotic resumption in mammalian oocytes. In addition, the phosphorylation sites of pCPEB1 were identified and are suggested to be phosphorylated by porcine Aurora A.     

An attempt to render oocytes and embryos free from the porcine circovirus type 2 after experimental in vitro exposure

The purpose of this study was to investigate the possibility of rendering oocytes and embryos free of porcine circovirus type 2 (PCV2). Groups of cumulus oocytes complexes, cumulus free oocytes, and embryos 3 to 5 d post breeding were exposed to PCV2 (10(5) TCID50/mL) prior to disinfection by washing and different combinations of enzymatic treatments. The study suggests that under the in vitro conditions used, standard washing procedures with, or without, trypsin or incorporating pronase or hyaluronidase and DNase/RNase in the treatment was not effective in rendering oocytes and embryos free from PCV2 nucleic acid. Since the virus is noncytopathic in cell culture and for embryonic cells, it appears that there is a possibility of introducing viral contamination through oocytes collected from infected pigs into the in vitro fertilization system with subsequent potential of producing in vitro fertilized embryos associated with PCV2.     

KIT-KIT ligand in the growth of porcine oocytes in primordial follicles

Mammalian ovaries are endowed with a huge number of small oocytes in primordial follicles (primordial oocytes). The mechanism regulating initiation of oocyte growth and follicular development is not well understood. Several growth factors and cytokines are known to be involved in oocyte growth and follicular development. Herein, the involvement of KIT, a receptor tyrosine kinase, and its ligand, KIT ligand (KL), in the initiation of porcine oocyte growth was examined. At first, KIT expression was examined immunohistochemically in primordial oocytes from neonatal (10-20 days) and prepubertal (about 6 months) pigs. Similar expression of KIT was detected in all oocytes from both the neonatal and prepubertal pigs. Next, to examine the growth of primordial oocytes, ovarian tissues containing primordial oocytes were xenotransplanted into immunodeficient SCID mice. Primordial oocytes from the neonatal pigs grew with follicular development as described previously, whereas those from the prepubertal pigs did not initiate growth in the xenografts after 2 months. To stimulate the growth of primordial oocytes from the prepubertal pigs, they were cultured in a medium supplemented with KL (50 and 100 ng/ml) for 1 or 3 days before xenografting. After 2 months, however, the oocytes did not grow, and the primordial follicles did not develop, although a higher number of primordial oocytes survived in the KL-treated tissues. These results suggest that KIT-KL might not be associated with the growth initiation of porcine primordial oocytes, although they do enhance the survival of the oocytes.     

Polo‐like kinase 1 inhibition results in misaligned chromosomes and aberrant spindles in porcine oocytes during the first meiotic division

Polo‐like kinase 1 (Plk1), a type of serine/threonine protein kinase, has been implicated in various functions in the regulation of mitotic processes. However, these kinase's roles in meiotic division are not fully understood, particularly in the meiotic maturation of porcine oocytes. In this study, the expression and spatiotemporal localization of Plk1 were initially assessed in the meiotic process of pig oocytes by utilizing Western blotting with immunofluorescent staining combined with confocal microscopy imaging technique. The results showed that Plk1 was expressed and exhibited a dynamic subcellular localization throughout the meiotic process. After germinal vesicle breakdown (GVBD), Plk1 was detected prominently around the condensed chromosomes and subsequently exhibited a similar subcellular localization to α‐tubulin throughout subsequent meiotic phases, with particular enrichment being observed near spindle poles at MI and MII. Inhibition of Plk1 via a highly selective inhibitor, GSK461364, led to the failure of first polar body extrusion in porcine oocytes, with the majority of the treated oocytes being arrested in GVBD. Further subcellular structure examination results indicated that Plk1 inhibition caused the great majority of oocytes with spindle abnormalities and chromosome misalignment during the first meiotic division. The results of this study illustrate that Plk1 is critical for the first meiotic division in porcine oocytes through its influence on spindle organization and chromosome alignment, which further affects the ensuing meiotic cell cycle progression.     

不同激活方法对猪体外成熟卵母细胞孤雌发育的影响

通过系统探讨不同孤雌激活方法对猪卵母细胞体外激活后发育效果的影响,比较了乙醇（Ethanol,EH）、离子霉素（Ionomycin,Ion：5μmol/L）、氯化锶（Strontium chloride hexahydrate,Sr^2＋：10 mmol/L）、6-二甲氨基嘌呤（6-dimethylaminopurine,6-DMAP：2 mmol/L）和放线菌酮（cycloheximide,CHX：10 mg/L）对猪卵母细胞激活发育的效果。结果表明：（1）9%EH激活处理10 min效果好于15 min;（2）使用9%EH激活处理10 min,再结合CHX、6-DMAP、Sr^2＋、CHX＋Sr^2＋、Sr^2＋＋6-DMAP、CHX＋6-DMAP或CHX＋6-DMAP＋Sr^2＋组处理3～4 h,以EH＋6-DMAP组效果最好,分裂率及囊胚率分别达到82.86%和22.86%;（3）在使用化学激活（Ion＋6-DMAP组和9%乙醇10 min＋6-DMAP组）和电激活（50 V/mm,50μs,2t）的方法中,Ion法激活猪卵母细胞效果较好,囊胚率达到37.50%;（4）卵母细胞包被的卵丘细胞层数不同对卵母细胞成熟激活有显著的影响,卵丘细胞层数4～6层和多于6层的卵丘-卵母细胞复合体孤雌激活的分裂率和囊胚率分别为（68.99%,32.56%）和（75.36%,37.68%）,2组之间差异不显著（P〉0.05）;但其显著高于其他组（P〈0.05）,这2组细胞在猪孤雌激活发育研究中是最佳的实验研究材料。     

Excess polyspermy reduces the ability of porcine oocytes to promote male pronuclear formation after in vitro fertilization

Male pronucleus (MPN) formation is a very important physiological event during fertilization, which affects in vitro production of transferrable embryos. The aim of this study was to find out the correlation between the number of penetrated sperm and the occurrence of failure of MPN formation in porcine oocytes. In vitro matured porcine oocytes were fertilized in vitro with frozen epididymal sperm. Two different frozen sperm lots were tested in this study, which were different in terms of polyspermy rates. The numbers and the status of penetrated sperm in oocytes were evaluated 10 h after insemination. Under high polyspermy condition, the polyspermy rate was 83.5% with an average mean of 3.5 sperms per penetrated oocyte, whereas the percentage of polyspermy was 65.5% with an average mean of 2.4 sperms per penetrated oocyte under moderate polyspermic condition. Correlation analysis revealed a negative correlation between the number of penetrated sperm and their MPN formation percentage both in the sperm lot of high polyspermy (R = −0.560, p < 0.05) and in the sperm lot of moderate polyspermy (R = −0.405, p < 0.05) which suggests that penetration of excessive spermatozoa disables the oocyte cytoplasm to promote MPN formation.     

Interlaboratory testing of porcine sera for antibodies to porcine circovirus type 2.

A panel of 20 porcine sera was distributed to 5 laboratories across Europe and Canada. Each center was requested to test the sera for the presence of porcine circovirus type 2 antibodies using the routine assays, indirect immunofluorescence assay (IFA) and indirect immunoperoxidase monolayer assay (IPMA), and to determine the titer of each serum. Results from all centers were then compiled and correlated. They demonstrate a wide variation in the titers obtained between laboratories. These differences were dependent on the assay used and the choice of fixative. In general, IPMA gave higher titers than did IFA, and paraformaldehyde gave higher titers than did acetone or ethyl alcohol. This report highlights the need for standardized procedures and biologicals for this virus.     

猪IL-2的原核表达及其促淋巴细胞增殖活性研究

为研究猪IL-2作为免疫佐剂的可行性,用IPTG诱导含有猪IL-2基因的BL21(DE3)LysS工程菌。目的蛋白经超声波破碎、洗涤、变性溶解后,用固化金属亲和层析法纯化后复性。MTT法对复性的重组蛋白的生物学活性进行分析。结果显示,相同浓度复性的IL-2与未复性的IL-2比较,刺激淋巴细胞体外增殖的能力差异极显著(P<0.01)。说明复性的重组猪IL-2具有良好的刺激淋巴细胞体外增殖的活性,在作为免疫佐剂方面具有潜在的应用价值。     

Effects of exposure to zearalenone on porcine oocytes and sperm during maturation and fertilization in vitro

The influence of acute exposure to zearalenone (ZEN) on porcine oocyte maturation, fertilization or sperm penetration ability during both in vitro maturation and fertilization was evaluated. First, oocytes were cultured in ZEN-containing (0-1000 μg/l) maturation medium and then fertilized. The oocytes maturing in vitro without ZEN were then fertilized in ZEN-containing fertilization medium. The maturation rates of oocytes and penetration ability of sperm decreased significantly in the presence of 1000 μg/l of ZEN. However, neither increases in the rates of degeneration and DNA fragmentation of oocytes nor reductions in normal and polyspermic fertilization were observed. ZEN did not affect the sperm penetration rates; however, 1000 μg/l ZEN had positive effects on normal and polyspermic fertilization rates. Therefore, it can be suggested that an acute exposure of porcine oocytes during maturation and of oocytes and sperm during fertilization to ZEN up to 1000 μg/l may not affect the fertility of the oocytes.     

Isolation of ovarian components essential for growth and development of mammalian oocytes in vitro

Mammalian ovaries contain a large number of oocytes, most of which degenerate either before or at various stages of growth. Dynamic and precise regulation in the ovary involves many factors, each with a unique role. Identifying the single most important factor is impossible; however, it may be possible to identify factors essential for oocyte growth. It is evident that oocytes can grow into competent ova in vitro; however, how faithfully the follicle should mimic the in vivo conditions remains unclear. In the culture system discussed in this review, bovine and mouse oocyte-granulosa cell complexes, at approximately the late mid-growth stage, spread on a substratum without the involvement of theca cells. The structural simplicity of this system is advantageous because it reduces the basic conditions essential for regulation of oocyte growth. Apart from biological factors, high concentrations of polyvinylpyrrolidone (molecular weight: 360000) improved oocyte growth. Among ovarian factors, androstenedione was used to compensate for the absence of theca cells, and it promoted both follicular growth and acquisition of oocyte meiotic competence. Most oocytes cultured in a group were viable after long-term culture, suggesting that unlike ovarian events, there was no exhaustive follicle selection. Collectively, oocytes and their associated granulosa cells can establish independent units capable of supporting oocyte growth in appropriately modified culture media.     

Supplementing media with NAD+ precursors enhances the in vitro maturation of porcine oocytes

In vitro maturation (IVM) is an important reproductive technology used to produce embryos in vitro. However, the developmental potential of oocytes sourced for IVM is markedly lower than those matured in vivo. Previously, NAD⁺-elevating treatments have improved oocyte quality and embryo development in cattle and mice, suggesting that NAD⁺ is important during oocyte maturation. The aim of this study was to examine the effects of nicotinic acid (NA), nicotinamide (NAM) and nicotinamide mononucleotide (NMN) on oocyte maturation and subsequent embryo development. Porcine oocytes from small antral follicles were matured for 44 h in a defined maturation medium supplemented with NA, NAM and resveratrol or NMN. Mature oocytes were artificially activated and presumptive zygotes cultured for 7 days. Additionally, oocytes were matured without treatment then cultured for 7 days with NMN. Supplementing the IVM medium with NA improved maturation and blastocyst formation while NAM supplementation improved cleavage rates compared with untreated controls. Supplementing the IVM or embryo culture media with NMN had no effect on maturation or embryo development. The results show that supplementing the maturation medium with NA and NAM improved maturation and developmental potential of porcine oocytes.     

Response to environmental temperatures in Brahman calves during the first compared to the second day after birth.

Brahman calves (n = 28) were used to evaluate the effect of environmental temperature during the 1st or 2nd d after birth. Calves were removed from their dams within 30 min of birth (newborn; D0) before suckling or at 20 h of age and fasted for 4 h before treatment (day-old; D1). Calves were placed in either a warm (W; 25 degrees C) or a cold (C; 5 degrees C) environment for 2 h and either maintained in or transferred to, respectively, W for 22 h. Blood samples were collected via jugular catheters at 15-min intervals beginning at initial placement in W or C through 3 h and at 4, 5, 6, 8, 10, 12, 14, 18, and 24 h. Rectal temperature (Tr) was recorded with each sample. Following the 60-min and 12-h samples, each calf was administered 1 liter of colostrum from its dam. Serum or plasma was analyzed for glucose, lactate, plasma urea nitrogen, triglycerides, nonesterified fatty acids, insulin, cortisol, triiodothyronine (T3), and thyroxine (T4). Rectal temperature of D0C calves was lower (P less than .05) than that of other calves from 75 min through 3 h. Insulin, lactate, T3, and plasma urea nitrogen concentrations were not different among all calves. Higher (P less than .01) cortisol and T4 concentrations were observed in D0 than in D1 calves. Cortisol (P less than .008) and nonesterified fatty acid (P less than .05) levels were greater in C than in W calves. All D0 calves had lower (P less than .0001) glucose concentrations than D1 calves until the 12-h feeding.(ABSTRACT TRUNCATED AT 250 WORDS)