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Find video protocols related to scientific articles indexed in Pubmed.
Expression and cellular distribution of estrogen and progesterone receptors and the real-time proliferation of porcine cumulus cells.
Zygote
PUBLISHED: 10-17-2014
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Summary Although the expression of estrogen and progesterone receptors within porcine ovary and cumulus-oocyte complexes (COCs) is well recognized, still little information is known regarding expression of the progesterone receptor (PGR), PGR membrane component 1 (PGRMC1) and of estrogen-related receptors (ERR? and ERR?/?) in separated cumulus cells in relation to real-time proliferation. In this study, a model of oocytes-separated cumulus cells was used to analyze the cell proliferation index and the expression PGR, PGRMC1 and of ERR? and ERR?/? during 96-h cultivation in vitro using real-time quantitative PCR (qRT-PCR) and confocal microscopic observation. We found that PGR protein expression was increased at 0 h, compared with PGR protein expression after 96 h of culture (P < 0.001). The expression of PGRMC1, ERR? and ERR?/? was unchanged. After using qRT-PCR we did not found statistical differences in expression of PGR, PGRMC1, ERR? and ERR?/? during 96 h of cumulus cells in vitro culture (IVC). We supposed that the differential expression of the PGR protein at 0 h and after 96 h is related to a time-dependent down-regulation, which may activate a negative feedback. The distribution of PGR, PGRMC1 proteins may be linked with the translocation of receptors to the cytoplasm after the membrane binding of respective agonists and intra-cytoplasmic signal transduction. Furthermore, cumulus cells analyzed at 0 h were characterized by decreased proliferation index, whereas those after 96 h of culture revealed a significant increase of proliferation index, which may be associated with differentiation/luteinization of these cells during real-time proliferation.
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Expression and cellular distribution of zona pellucida glycoproteins in canine oocytes before and after in vitro maturation.
Zygote
PUBLISHED: 10-16-2014
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Summary This study was aimed at investigating zona pellucida glycoproteins (ZP) ZP2, ZP3 mRNA expression as well as ZP3, ZP4 (ZPB) protein distribution before and after in vitro maturation (IVM) in canine oocytes. The cumulus-oocyte complexes (COCs) were recovered from 27 anoestrous mongrel bitches and matured for 72 h in TCM199 medium. The canine COCs were analysed before and after IVM. Using real-time quantitative polymerase chain reaction (RQ-PCR), both groups of oocytes were analysed for detection of ZP2 and ZP3 mRNA profiles as well as using confocal microscopic analysis for observation of ZP3 and ZP4 protein distribution. In post-IVM canine oocytes an increase in transcript content of ZP2 and ZP3 genes as well as a decrease in ZP3 and ZP4 protein levels were observed when compared with pre-IVM oocytes. Moreover, the ZP4 protein before IVM was significantly distributed in the peripheral area of cytoplasm, whereas after IVM it was localized rather than in the entire cytoplasm. In contrast, the ZP3 protein was found both before and after IVM was distributed in the peripheral area of the cytoplasm. In conclusion, we suggest that the expression of ZP2 and ZP3 genes is associated with the maturation stage of canine oocytes, as higher mRNAs levels were found after IVM. However, a decreased expression of ZP3 and ZP4 proteins after IVM suggests maturation-dependent down-regulation of these protein translations, which may result in disturbed fertilization.
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Is the function of the porcine sperm reservoir restricted to the ovulatory period?
J. Reprod. Dev.
PUBLISHED: 06-24-2014
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The uterotubal junction (UTJ) and caudal isthmus are recognized as a functional pre-ovulatory sperm reservoir (SR). Spermatozoa are released from the SR in a complex and concerted action. However, whether this functionality is restricted only to the ovulatory period is still open to debate. Our study was aimed to analyze the presence of spermatozoa within the UTJ (SR), isthmus (ISTH) and ampulla (AMP) after laparoscopic intrauterine insemination (LIUI) either in the peri- (PERI) or post-ovulatory (POST) period or at mid cycle (MID). Each uterine horn of estrus synchronized gilts (n=12) was inseminated with 20 ml sperm (29.5×10(6) cells/ml). Oviducts were recovered 7 h after LIUI and separated into the UTJ, ISTH and AMP, and sections were flushed with 10 ml PBS+EDTA solution. After centrifugation, the sperm pellet was evaluated by ?e?ovský staining. The median sperm numbers in the PERI, POST and MID groups were 578, 171 and 789 in the UTJ; 545, 233 and 713 in the ISTH; and 496, 280 and 926 in the AMP, respectively, and there were differences between the POST and MID groups (P<0.05) but not between the oviductal sections of each group (P>0.05). Compared with the MID group, the percent of intact sperm cells was higher (P<0.01) in the PERI and POST groups (32.8 vs. 66.4 and 76.8%). Also, the percentages of aberrations in the acrosome and tail were higher (P<0.05) in the MID group. Based on this, it can be assumed that the sperm reservoir is active during different phases of the estrus cycle. However, the mid-cycle oviduct environment considerably impairs sperm cell quality.
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Low and High Dietary Protein:Carbohydrate Ratios during Pregnancy Affect Materno-Fetal Glucose Metabolism in Pigs.
J. Nutr.
PUBLISHED: 12-18-2013
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Inadequate dietary protein during pregnancy causes intrauterine growth retardation. Whether this is related to altered maternal and fetal glucose metabolism was examined in pregnant sows comparing a high-protein:low-carbohydrate diet (HP-LC; 30% protein, 39% carbohydrates) with a moderately low-protein:high-carbohydrate diet (LP-HC; 6.5% protein, 68% carbohydrates) and the isoenergetic standard diet (ST; 12.1% protein, 60% carbohydrates). During late pregnancy, maternal and umbilical glucose metabolism and fetal hepatic mRNA expression of gluconeogenic enzymes were examined. During an i.v. glucose tolerance test (IVGTT), the LP-HC-fed sows had lower insulin concentrations and area under the curve (AUC), and higher glucose:insulin ratios than the ST- and the HP-LC-fed sows (P < 0.05). Insulin sensitivity and glucose clearance were higher in the LP-HC sows compared with ST sows (P < 0.05). Glucagon concentrations during postabsorptive conditions and IVGTT, and glucose AUC during IVGTT, were higher in the HP-LC group compared with the other groups (P < 0.001). (13)C glucose oxidation was lower in the HP-LC sows than in the ST and LP-HC sows (P < 0.05). The HP-LC fetuses were lighter and had a higher brain:liver ratio than the ST group (P < 0.05). The umbilical arterial inositol concentration was greater in the HP-LC group (P < 0.05) and overall small fetuses (230-572 g) had higher values than medium and heavy fetuses (?573 g) (P < 0.05). Placental lactate release was lower in the LP-HC group than in the ST group (P < 0.05). Fetal glucose extraction tended to be lower in the LP-HC group than in the ST group (P = 0.07). In the HP-LC and LP-HC fetuses, hepatic mRNA expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC) was higher than in the ST fetuses (P < 0.05). In conclusion, the HP-LC and LP-HC sows adapted by reducing glucose turnover and oxidation and having higher glucose utilization, respectively. The HP-LC and LP-HC fetuses adapted via prematurely expressed hepatic gluconeogenic enzymes.
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Saving genetic resources of native pigs in occidental and Oriental countries - practical examples of the characterization and utilization of native pigs in Hungary and Laos.
J. Reprod. Dev.
PUBLISHED: 10-29-2013
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Worldwide, only a few "fatty" pig breeds exist with different and/or regional utilization. Using the Hungarian Mangalica, which almost went extinct in Europe and the Lao Moo Lat pig, which still has a large population in South-East Asia as exemples, we wanted to demonstrate that indigenous (fatty) pig breeds may represent both national value and tremendous economic potential. Since these less prolific and less productive breeds cannot contribute to mass production, new market roles and methods should be established for them in the premium segment of pork trading. Thus their preservation and propagation needs the comprehensive collaboration of commercial, governmental actors and researchers. Briefly summarizing the history, we report the current results of reproductive physiology research. The commercial renaissance of Mangalica pigs is indebted to the enthusiastic efforts of basic scientists, pig breeding experts and dedicated Mangalica producers. Scientific achievements were applied to practical breeding and production of delicious pork and processed products, which ultimately made the economic success in the Mangalica sector possible. Both, research on and utilization of endangered (pig) breeds maintain not only breed diversities, but also may improve the livelihood of farmers worldwide.
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Expression of INH?A and INH?B proteins in porcine oocytes cultured in vitro is dependent on the follicle size.
Zygote
PUBLISHED: 10-19-2013
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Summary The current study aimed to investigate differential expression of inhibin ?A (INH?A) and inhibin ?B (INH?B) in porcine oocytes before or after in vitro maturation (IVM) isolated from follicles of various sizes. Porcine oocytes isolated from large, medium and small follicles (40 from each) were used to study the INH?A and INH?B protein expression pattern using western blot analysis before or after 44 h of oocyte IVM. An increased expression of INH?A was found in oocytes collected from large and medium follicles compared with small follicles before or after IVM (P < 0.001, P < 0.05, respectively). Similarly, higher INH?B levels were observed in oocytes recovered from large follicles compared with small (P < 0.01). As INH?A and INH?B are expressed in both porcine follicular somatic cells and oocytes, it can be assumed that these transforming growth factor beta (TGF?) superfamily factors are involved in the regulation of molecular bi-directional pathways during follicle and oocyte development, and can be recognized as markers of follicle and oocyte maturation. Moreover, the current study clearly demonstrated that inhibin expression is substantially associated with porcine follicle growth and development.
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Short-term cultivation of porcine cumulus cells influences the cyclin-dependent kinase 4 (Cdk4) and connexin 43 (Cx43) protein expression--a real-time cell proliferation approach.
J. Reprod. Dev.
PUBLISHED: 05-17-2013
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The CC (cumulus cell) proliferation index in relation to the expression and distribution of Cdk4 and Cx43 proteins, which are crucial factors for oocyte maturation, was investigated. Cumulus-oocyte complexes (COCs) were recovered from pubertal crossbred Landrace gilts and treated with collagenase, and separated CCs were cultured in standard TCM199 medium for 44 h. At each step of in vitro cultivation (IVC) of CCs (0, 12, 24 and 44 h), a normalized proliferation index was assessed. Cdk4 and Cx43 protein expression and the CC-specific cellular distribution were analyzed by confocal microscopic observation. The normalized proliferation index (number of cells attached, measured by impedance) was increased in the first 12 h of IVC (P<0.01) and differed between 12 h and 24 h of cultivation (P<0.001). Later, between 24 h-44 h of IVC, the CC proliferation rate was stable, and no significant differences were observed. Based on the confocal microscopic observation, increased expression of both Cdk4 and Cx43 was found after 44 h of IVC compared with the expression of these proteins before IVC. Moreover, after IVC, a substantial translocation of Cdk4 and Cx43 was noted from the nucleus to the cytoplasm of CCs. In conclusion, it was demonstrated for the first time that CCs can be cultured in vitro separately without oocytes and that the proliferation index was significantly increased in the first 12 h of IVC, which may reflect the process of ordinary cumulus cell expansion. Furthermore, the expression of both Cdk4 and Cx43 in CCs suggested that these proteins may be regarded as markers not only of proper oocyte maturation but also of CC differentiation. Translocation of these proteins into the cytoplasm of CCs after 44 h of IVC may be related to the expansion process.
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Differential expression of GDF9, TGFB1, TGFB2 and TGFB3 in porcine oocytes isolated from follicles of different size before and after culture in vitro.
Acta Vet. Hung.
PUBLISHED: 02-27-2013
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The TGFB superfamily genes are involved in several important cell functions, including proliferation and differentiation, and the role of the expression of these genes in growth and development of theca and granulosa cells is well recognised. However, the dependence between the stage of oocyte maturation or follicular size and the expression of these genes in pigs is still not entirely known. This study was aimed at investigating the expression pattern of GDF9, TGFB1, TGFB2 and TGFB3 in porcine oocytes before and after in vitro maturation (IVM) as well as in oocytes collected from follicles of different sizes. RQ-PCR was performed to analyse the expression of GDF9, TGFB1, TGFB2 and TGFB3 in oocytes before and after IVM (oocytes cultured for 44 h in TCM-199), isolated from large (> 5 mm), medium (3-5 mm) and small (< 3 mm) follicles collected from ovaries of 28 puberal crossbred Landrace gilts after slaughter. We found an increased expression of both TGFB1 and TGFB2 in oocytes before IVM collected from large as compared to medium and small follicles (P < 0.05, P < 0.001, P < 0.01, P < 0.05, respectively). In these groups of oocytes we did not observe differences in GDF9 and TGFB3 mRNA levels. However, after IVM, GDF9 protein distribution in oocytes was significantly higher in large and medium follicles as compared to small ones (P < 0.01, P < 0.001, respectively). Moreover, an increased TGFB1, TGFB2 and TGFB3 proteins pattern was observed in oocytes of large compared to small follicles. The highest GDF9 and TGFB1 mRNA levels were found in oocytes after IVM compared to those before IVM. Based on our study we can suppose that the distribution pattern of TGFB superfamily genes is associated with the stage of maturation of porcine oocytes and the follicle size. Furthermore, GDF9 and TGFB1 may serve as molecular markers of the develop-mental potential of porcine oocytes. The confocal microscopic observation revealed that TGFB1 and TGFB3 were translocated between the zona and the cytoplasm of oocytes, depending on the stage of maturation and follicle size.
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The Akt/mTor signaling cascade is modified during placentation in the porcine uterine tissue.
Reprod Biol
PUBLISHED: 02-07-2013
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Recently we showed that essential components for the initiation of protein synthesis, namely the eukaryotic initiation factor 4E (eIF4E, mRNA-cap-binding protein) and its repressors 4E-BP1 as well as 4E-BP2, are proteolytically processed in the porcine endometrium during implantation. Here, the situation during placentation was compared with ovariectomized (OVX) animals and animals on pregnancy day 1 (PD1). Furthermore, the research was extended to factors which phosphorylate eIF4E and 4E-BPs and regulate their activities. These are the protein kinase B/mammalian target of rapamycin kinase (Akt/mTor) with the regulators Raptor and Rictor as well as the mitogen activated protein kinases (MAPKs): extra cellular-signal regulated kinase 1 and 2 (ERK1 and ERK2). Striking differences in the placentation site (PS) and the areas aside from PS (peri-PS) were observed. EIF4E and 4E-BP2 truncation as well as 4E-BP1 degradation took place in the endometrium of the peri-PS on PD24. Accompanied by a fragmentation of Akt/mTor, no expression of Rictor was observed, whereas the abundance of Raptor was not altered. On the contrary, MAPKs expression and phosphorylation remained almost stable in the peri-PS. In conclusion, the results indicated that on PD24 the translational regulation was shifted to 4E-BP2 control. Furthermore, the Akt/mTor signaling cascade seemed to be down regulated which suggest reduced phosphorylation of 4E-BP2. Whereas Akt was proteolyzed, the observed mTor fragments represented most likely splicing variants. The results indicate that translational control of gene expression is an important feature in the porcine endometrium during early pregnancy.
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Real-time proliferation of porcine cumulus cells is related to the protein levels and cellular distribution of Cdk4 and Cx43.
Theriogenology
PUBLISHED: 01-31-2013
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The proper maturation of cumulus somatic cells depends on bidirectional communication between the oocyte and the surrounding cumulus cells (CCs). The aim of this study was (i) to investigate maturation markers, such as Cx43 and Cdk4 protein levels, and (ii) to analyze the distribution of these two proteins in CCs cultured for 44, 88, 132, and 164 hours in both separated and cumulus-enclosed oocyte cultures. CCs were isolated from porcine ovarian follicles after the treatment of the recovered COCs with collagenase. Then, the separated CCs were cultured in TCM-199 for 0 to 164 hours, using a real-time cellular analyzer; however, the immunostaining was performed only after 44, 88, and 132 hours. The protein levels and distribution were analyzed using confocal microscopy. After the CCs underwent in vitro cultivation (IVC) for 25 hours, a logarithmically increasing normalized proliferation index was found throughout the entire 164 hours cultivation time. The Cx43 and Cdk4 proteins were observed at higher levels after 44 hours of culture than before IVC. After 88 and 132 hours of IVC, no significant alterations in either mRNA or protein levels of Cx43 and Cdk4 were found. Cx43 and Cdk4 were localized in the cell nucleus before IVC, whereas after 44, 88, and 132 hours of IVC, both proteins translocated to the cytoplasm. In cumulus-enclosed oocyte cultures, Cdk4 was localized both in the nucleus and cytoplasm, whereas Cx43 was only in the cytoplasm. Additionally, only low levels of the cumulus expansion markers MIS and SNAT3 were observed. In summary, we could demonstrate that the in vitro cultivation of CCs was associated with cell proliferation and that Cx43 and Cdk4 gene expression was upregulated after IVC, resulting in significantly higher protein levels. Moreover, the two proteins translocated from the nucleus to the cytoplasm of the CCs during IVC. The protein distribution is presumably related to different protein functions during bidirectional communication via gap junction communication.
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4E-BP1 degradation and eIF4E truncation occur spatially distinctly in the porcine uterine epithelia and are features of noninvasive implantation in the pig.
Mol. Reprod. Dev.
PUBLISHED: 05-24-2011
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The implantation of the blastocyst into the endometrium is an indispensable premise for successful embryonic development. This process is regulated by maternal and embryonic signals that influence gene expression at the translational level, among other processes. Recently, we have shown that proteolytical cleavage of the prototypical 25-kDa, mRNA cap-binding protein eIF4E produces a stable variant with a molecular mass of approximately 23?kDa exclusively in the porcine endometrium during implantation. This is accompanied by dephosphorylation and reduction of the abundant repressor 4E-BP1. Here, we investigate the distribution of the truncated eIF4E and of 4E-BP1 in the porcine uterine tissue, their binding in native samples, and we analyzed eIF4E-, eIF4G-, and 4E-BP1-specific proteolytic activities. Our results show that in pigs, the truncated eIF4E is located in the endometrial luminal epithelium during implantation. Neither glandulary tissue nor stroma expressed any truncated eIF4E. The reduced abundance of 4E-BP1 during implantation is mainly the result of decay in the glandular epithelia. Moreover, steroid replacements, in vitro protease assays, and cell lysate fractionation showed that eIF4E cleavage and 4E-BP1 decay both depended on the ovarian steroid hormones estradiol and progestrone, but these effects are the result of different proteolytic activities. Although eIF4G cleavage also depends on calcium, stimulation by these steroids could not be established. We propose that the translation initiation process in the endometrium is differently regulated by the truncated eIF4E, utilizing different abundances of 4E-BP1 and binding dynamic of eIF4E/4E-BP1 in distinct forms of implantation.
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Sperm migration in pigs after deep intrauterine and intraperitoneal insemination.
J. Reprod. Dev.
PUBLISHED: 01-18-2011
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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.
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Endocrine effects of GnRH agonist application to early pregnant gilts.
J. Reprod. Dev.
PUBLISHED: 12-09-2010
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The hypothesis of the present study was that a GnRH agonist application at early pregnancy would alter the pattern of the key reproductive hormones LH and FSH, and subsequently that of estradiol (E2) and especially progesterone (P4), and improve the conditions for embryo survival in early pregnant gilts. Therefore, the endocrine effects of a GnRH agonist (GnRHa) application to gilts (n=11 GnRHa treated, n=9 saline Controls) were studied in blood samples from the Vena cava caudalis. GnRHa injected on Day 12 after insemination induced elevated (P<0.01) LH and FSH levels for at least 180 min. However, subsequent LH concentrations were not altered up to Day 21 of pregnancy. LH pulse number, estimated in 6-h period samples on Days 13, 15 and 17, was not influenced by treatment and pregnancy. LH pulse amplitude was decreased (P<0.05) on Days 13 to 17 in pregnant gilts of both groups, but not in nonpregnant animals. In pregnant GnRHa-treated gilts, the basal LH level was elevated compared with the Controls (P<0.01). Additionally, differences (P<0.05) in basal LH were present between the pregnant and nonpregnant animals. The P4 and E2 secretion pattern was not affected by GnRHa. P4 concentrations increased (P<0.01) from Day 10 to Day 14 regardless of the treatment. P4 revealed a pulse-like pattern, but without a definite relation to the LH pulse characteristics. Also, pregnancy rate (73 vs. 67%) and the number of fetuses (12.8 ± 2.3 vs. 11.6 ± 2.3) were unaffected in the treated and Control gilts, respectively. The present study did not confirm the initial hypothesis that a GnRHa-mediated LH effect could alter ovarian steroid secretion and favorably support early embryo development and pregnancy outcome.
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Natural occurrence and physiological role of a truncated eIF4E in the porcine endometrium during implantation.
Biochem. J.
PUBLISHED: 09-22-2010
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The present study is the first report providing evidence for a physiological role of a truncated form of the mRNA cap-binding protein eIF4E1 (eukaryotic initiation factor 4E1). Our initial observation was that eIF4E, which mediates the mRNA cap function by recruiting the eIF4F complex (composed of eIF4E, 4G and 4A), occurs in two forms in porcine endometrial tissue in a strictly temporally restricted fashion. The ubiquitous prototypical 25 kDa form of eIF4E was found in ovariectomized and cyclic animals. A new stable 23 kDa variant, however, is predominant during early pregnancy at the time of implantation. Northern blotting, cDNA sequence analysis, in vitro protease assays and MS showed that the 23 kDa form does not belong to a new class of eIF4E proteins. It represents a proteolytically processed variant of eIF4E1, lacking not more than 21 amino acids at the N-terminus. Steroid replacements indicated that progesterone in combination with 17?-oestradiol induced the formation of the 23 kDa eIF4E. Modified cell-free translation systems mimicking the situation in the endometrium revealed that, besides eIF4E, eIF4G was also truncated, but not eIF4A or PABP [poly(A)-binding protein]. The 23 kDa form of eIF4E reduced the repressive function of 4E-BP1 (eIF4E-binding protein 1) and the truncated eIF4G lacked the PABP-binding site. Thus we suggest that the truncated eIF4E provides an alternative regulation mechanism by an altered dynamic of eIF4E/4E-BP1 binding under conditions where 4E-BP1 is hypophosphorylated. Together with the impaired eIF4G-PABP interaction, the modified translational initiation might particularly regulate protein synthesis during conceptus attachment at the time of implantation.
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Meiotic progression, mitochondrial features and fertilisation characteristics of porcine oocytes with different G6PDH activities.
Reprod. Fertil. Dev.
PUBLISHED: 05-11-2010
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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.
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Influence of synthetic lamprey GnRH-III on gonadotropin release and steroid hormone levels in gilts.
Theriogenology
PUBLISHED: 04-27-2010
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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 ?g Maprelin® XP10 (lGnRH-III, group Maprelin, n = 6), 50 ?g 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 ?g Maprelin® XP10 (n = 3), 50 ?g 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.
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On the effects of the Fusarium toxin deoxynivalenol (DON) administered per os or intraperitoneal infusion to sows during days 63 to 70 of gestation.
Mycotoxin Res
PUBLISHED: 02-26-2010
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Six pregnant sows of 180.6?±?5.6 kg were fed either a Fusarium-contaminated (4.42 mg DON and 48.3 µg ZON per kg, DON per os, n?=?3) or a control diet (0.15 mg DON and 5 µg ZON/kg) in the period of days 63 and 70 of gestation. On day 63 of gestation, sows fed the control diet were implanted with an intraperitoneal osmotic minipump (delivery rate of 10 µL/h, for 7 days) containing 50 mg pure (98%) DON in 2 ml 50% DMSO (DON ip, n?=?3). Frequent plasma samples were taken to estimate the kinetics after oral and ip DON exposure. The intended continuous delivery of DON by the intraperitoneal minipump could not be shown, as there was a plasma peak (Cmax) of 4.2-6.4 ng DON/mL either immediately (sow IP-2+3) or 2.5 h (sow IP-1) after implantation of the pump followed by a one-exponential decline with a mean half-time (t1/2) of 1.75-4.0 h and only negligible DON plasma concentrations after 12 h. Therefore, the DON ip exposure has to be regarded as one single dose 1 week before termination of experiment. The DON per os sows showed a mean basis level (after achieving a steady state) of DON plasma concentration of about 6-8 ng/mL, as also indicated by the plasma DON concentration at the termination of the experiment. On day 70, caesarean section was carried out, the fetuses were killed immediately after birth, and samples of plasma, urine, and bile were taken to analyze the concentration of DON and its metabolite de-epoxy-DON. At necropsy there were no macroscopic lesions observed in any organ of either sows or piglets. Histopathological evaluation of sows liver and spleen revealed no alterations. The proliferation rate of peripheral blood mononuclear cells (PBMC) with or without stimulation was not affected by the kind of DON treatment. The exposure of pregnant sows at mid-gestation (days 63-70, period of organogenesis) to a Fusarium toxin-contaminated diet (4.42 mg DON and 0.048 mg ZON per kg) or pure DON via intraperitoneal osmotic minipump did not cause adverse effects on health, fertility, maintenance of pregnancy, and performance of sows and their fetuses. However, DON was detected in fetus plasma, indicating that this toxin can pass the placental barrier and may cause changes in the proportion of white blood cells (lower monocyte and neutrophil and higher lymphocyte proportion in DON per os fetuses).
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Follicular size is associated with the levels of transcripts and proteins of selected molecules responsible for the fertilization ability of oocytes of puberal gilts.
J. Reprod. Dev.
PUBLISHED: 08-11-2009
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The maturation and developmental competence of the oocyte is acquired during folliculogenesis. It is still unclear whether follicle size is associated with the levels of transcript and protein encoding molecules contributing to the fertilization ability of the porcine oocyte. Follicles were dissected from porcine ovaries after slaughter and classified as small (< 3 mm), medium (3-5 mm) or large (>5 mm), aspirated cumulus-oocyte complexes were cultured in standard porcine IVM culture medium (TCM 199) for 44 h. In developmentally competent oocytes, assessed by determining the activity of glucose-6-phosphate dehydrogenase (G6PDH) using a brilliant cresyl blue (BCB) test, real-time quantitative PCR reaction methods, western-blot and confocal microscopy analysis were applied to determine the transcript levels of porcine zona pellucida glycoproteins pZP1, pZP2, pZP3, pZP3 alpha and integrins beta 1 and beta 2, as well as the levels of pZP3 and integrin beta 2 proteins. We observed significantly higher levels of pZP1, pZP3 and integrin beta1 and beta2 transcripts in oocytes collected from medium follicles as compared with small follicles (P<0.001). Moreover, we found an increased content of all investigated mRNAs in oocytes isolated from large follicles as compared with small follicles (P<0.001). Western-blot analysis demonstrated a higher level of pZP3 protein in oocytes isolated from large and medium follicles as compared with small follicles (P<0.001). Our results suggest that the levels of transcripts and proteins for selected molecules contributing to the fertilization ability of oocytes are associated with follicular size in puberal gilts.
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High and low protein? carbohydrate dietary ratios during gestation alter maternal-fetal cortisol regulation in pigs.
PLoS ONE
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Imbalanced maternal nutrition during gestation can cause alterations of the hypothalamic-pituitary-adrenal (HPA) system in offspring. The present study investigated the effects of maternal low- and high-protein diets during gestation in pigs on the maternal-fetal HPA regulation and expression of the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11?-hydroxysteroid dehydrogenase 1 and 2 (11?-HSD1 and 11?-HSD2) and c-fos mRNAs in the placenta and fetal brain. Twenty-seven German Landrace sows were fed diets with high (HP, 30%), low (LP, 6.5%) or adequate (AP, 12.1%) protein levels made isoenergetic by varying the carbohydrate levels. On gestational day 94, fetuses were recovered under general anesthesia for the collection of blood, brain and placenta samples. The LP diet in sows increased salivary cortisol levels during gestation compared to the HP and AP sows and caused an increase of placental GR and c-fos mRNA expression. However, the diurnal rhythm of plasma cortisol was disturbed in both LP and HP sows. Total plasma cortisol concentrations in the umbilical cord vessels were elevated in fetuses from HP sows, whereas corticosteroid-binding globulin levels were decreased in LP fetuses. In the hypothalamus, LP fetuses displayed an enhanced mRNA expression of 11?-HSD1 and a reduced expression of c-fos. Additionally, the 11?-HSD2 mRNA expression was decreased in both LP and HP fetuses. The present results suggest that both low and high protein?carbohydrate dietary ratios during gestation may alter the expression of genes encoding key determinants of glucocorticoid hormone action in the fetus with potential long-lasting consequences for stress adaptation and health.
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Expression and cellular distribution of INHA and INHB before and after in vitro cultivation of porcine oocytes isolated from follicles of different size.
J. Biomed. Biotechnol.
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Cumulus-oocyte-complexes (COCs) were collected from small (<3?mm), medium (3-5?mm), and large (>5?mm) porcine follicles, and the INHA and INHB expression and cellular localization were studied. Developmentally competent (BCB+) COCs were cultured for 44?h. Samples of mRNA were isolated before and after in vitro maturation (IVM) from oocytes collected from follicles of different size for RQ-PCR assay. The INHA and INHB protein distribution within the oocytes was observed by confocal microscopy. INHA mRNA expression was increased in oocytes from large compared to medium and small follicles before IVM (P < 0.001), and to oocytes of small follicles after IVM (P < 0.001). The INHB expression was not different before IVM, but the IHNB mRNA level was gradually higher in oocytes from large follicles after IVM (P < 0.01). INHA was not differently expressed before IVM; however, in large follicle oocytes the protein was distributed in the peripheral area of the cytoplasm; in oocytes from small follicles it was in the entire cytoplasm. After IVM, INHA was strongly expressed in oocytes from small follicles and distributed particularly in the zona pellucida (ZP). Similarly and both before and after IVM, INHB protein was highly expressed in small follicle oocytes and within the cytoplasm. In summary, INHs can be recognized as a marker of porcine oocyte quality.
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The eIF4E repressor protein 4E-BP2 is merely truncated, despite 4E-BP1 degradation in the porcine uterine tissue during implantation.
Mol. Reprod. Dev.
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Recently, we identified an N-terminally truncated form of the mRNA cap binding protein eIF4E in the porcine luminal epithelium during implantation. EIF4E truncation is accompanied by degradation of the eIF4E-repressor protein 4E-BP1. In this study, we investigated whether or not the other members of the eIF4E-repressor family, namely 4E-BP2 and 4E-BP3, were also modified during early pregnancy. We did not detect 4E-BP3 in the uterine tissue; however, 4E-BP2 emerged in one or two stable fragments on pregnancy day 15. 4E-BP2 truncation most likely occurs at the N-terminus, and this calcium-stimulated processing depends on progesterone and estradiol. The activities targeting eIF4E, 4E-BP1, and 4E-BP2 were found in different fractions after anionic exchange chromatography, indicating the action of different proteases. Detailed protein interaction studies with immobilized anti-eIF4E and m(7) GTP-Sepharose showed a differential binding of the 4E-BP2 isoforms to the eIF4E variants and to the cap structure. In general, truncation of eIF4E reduces the inhibitory impact of 4E-BP2, whereas truncation of 4E-BP2 restores repression by binding the prototype eIF4E. In this context, we suggest long-term translational repression by the truncated 4E-BP2 is affected by the loss of the RAIP motif located at the N-terminus, which is indispensable for phosphorylation and deactivation of the molecule. In conclusion, we propose a tightly balanced regulation of the truncation of the cap-binding complex component eIF4F and degradation of 4E-BP1 and/or truncation of 4E-BP2 that together ensures correct translational control during the dynamic process of conceptus implantation.
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High-protein-low-carbohydrate diet during pregnancy alters maternal plasma amino acid concentration and placental amino acid extraction but not fetal plasma amino acids in pigs.
Br. J. Nutr.
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A high protein-low-carbohydrate diet during pregnancy can cause intra-uterine growth restriction. However, its impact during pregnancy on maternal, umbilical and fetal plasma amino acid (AA) profiles is unknown. A maternal high-protein (30 %)-low-carbohydrate (HP-LC) diet was compared with isoenergetic standard (12·1 % crude protein; ST) and low-protein (6·5 %)-high-carbohydrate (LP-HC) diets fed to nulliparous pregnant sows to examine changes in AA concentrations in maternal, venous and arterial umbilical and fetal plasma in mid and late pregnancy. At 64 and 94 days of pregnancy (dp), sows underwent Caesarean section, and maternal, umbilical and fetal plasma samples were collected. The HP-LC diet mainly affected maternal plasma AA concentrations. Plasma concentrations of Ile and Val were increased and those of Ala, Glu and Gly were decreased (P ? 0·05) in HP-LC compared with ST sows at 64 and 94 dp. The LP-HC diet decreased fetal plasma Glu concentration compared with the ST diet at 94 dp. Substantial AA catabolism was reflected by increased (P ? 0·05) maternal and fetal plasma urea concentrations with the HP-LC compared with the ST and LP-HC diets at 94 dp. Fractional placental extraction of Val was higher whereas those of Ala, Gln and Glu were lower in the HP-LC compared with the ST sows at 64 and 94 dp (P ? 0·05). Reduced fetal mass at 94 dp was accompanied by reduced fetal extraction of Lys and Pro in the HP-LC group (P ? 0·05). In conclusion, a maternal HP-LC diet during pregnancy altered maternal plasma composition of many AA and modified placental AA extraction to compensate for imbalanced maternal nutrient intake.
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Intrauterine growth retarded progeny of pregnant sows fed high protein:low carbohydrate diet is related to metabolic energy deficit.
PLoS ONE
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High and low protein diets fed to pregnant adolescent sows led to intrauterine growth retardation (IUGR). To explore underlying mechanisms, sow plasma metabolite and hormone concentrations were analyzed during different pregnancy stages and correlated with litter weight (LW) at birth, sow body weight and back fat thickness. Sows were fed diets with low (6.5%, LP), adequate (12.1%, AP), and high (30%, HP) protein levels, made isoenergetic by adjusted carbohydrate content. At -5, 24, 66, and 108 days post coitum (dpc) fasted blood was collected. At 92 dpc, diurnal metabolic profiles were determined. Fasted serum urea and plasma glucagon were higher due to the HP diet. High density lipoprotein cholesterol (HDLC), %HDLC and cortisol were reduced in HP compared with AP sows. Lowest concentrations were observed for serum urea and protein, plasma insulin-like growth factor-I, low density lipoprotein cholesterol, and progesterone in LP compared with AP and HP sows. Fasted plasma glucose, insulin and leptin concentrations were unchanged. Diurnal metabolic profiles showed lower glucose in HP sows whereas non-esterified fatty acids (NEFA) concentrations were higher in HP compared with AP and LP sows. In HP and LP sows, urea concentrations were 300% and 60% of AP sows, respectively. Plasma total cholesterol was higher in LP than in AP and HP sows. In AP sows, LW correlated positively with insulin and insulin/glucose and negatively with glucagon/insulin at 66 dpc, whereas in HP sows LW associated positively with NEFA. In conclusion, IUGR in sows fed high protein:low carbohydrate diet was probably due to glucose and energy deficit whereas in sows with low protein:high carbohydrate diet it was possibly a response to a deficit of indispensable amino acids which impaired lipoprotein metabolism and favored maternal lipid disposal.
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Laparoscopy guided Doppler ultrasound measurement of fetal blood flow indices during early to mid-gestation in pigs.
J. Reprod. Dev.
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The objectives of this study were to obtain relevant blood flow indices of umbilical arteries (UmA) of porcine fetuses using a laparoscopic ultrasound probe and to relate these data with fetal size at early to mid gestation. Fetal parameters and flow indices, i.e., fetal length and area, fetal heart rate (FHR), systolic pulse duration (T1), interpulse duration (T2), T2/T1 ratio, peak systolic velocity (PSV), time averaged velocity (TAV), resistance index (RI) and pulsatility index (PI), were measured in 182 fetuses of 26 pregnant Landrace gilts on pregnancy day (PD) 36 (122 fetuses from 17 gilts), PD42 (19 fetuses from 3 gilts) and PD51 (42 fetuses from 6 gilts). Fetal heart rate was higher on PD36 than on PD42 (P<0.05). No differences (P>0.05) were obtained concerning systolic pulse duration, flow velocities and RI. On PD42, the PI was lower (P<0.05), while the interpulse duration (P=0.06) and T2/T1 ratio tended (P=0.08) to be higher on PD42 compared with PD36 and to PD51. To find differences in UmA blood flow parameters concerning fetal size, i.e., fetal length, fetuses were retrospectively grouped as follows: small (lower 25%), medium (mean 50%) and large (upper 25%), respectively. Although, fetuses differed in size (P<0.001) within and between days of pregnancy, FHR, PSV, TAV, RI and PI did not differ (P>0.05) among the size classes. Only systolic pulse duration tended to be longer (P=0.05) in large compared with small fetuses on PD36, and interpulse duration was lower in large fetuses on PD36 in comparison with PD51 (P<0.05). Though there was no link between fetal blood flow indices and fetal intrauterine growth retardation (IUGR), with further studies based on these flow indices, it might be possible to evaluate nutrient- or stress-related influences on fetal growth and development, particularly in the case of IUGR.
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Expression and cellular distribution of cyclin-dependent kinase 4 (Cdk4) and connexin 43 (Cx43) in porcine oocytes before and after in vitro maturation.
Acta Vet. Hung.
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It is recognised that connexin 43 (Cx43) and cyclin-dependent kinase 4 (Cdk4) are involved in the cumulus cell-oocyte communication via gap junctions and the control of cell cycle progress. However, little is known about their mRNA expression pattern and encoded proteins distribution in porcine oocytes during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were collected from 31 puberal crossbred Landrace gilts and analysed for their Cdk4 and Cx43 mRNA expression using RQ-PCR and for the respective protein expression by confocal microscopic observations. An increased Cdk4 and Cx43 mRNA expression was found in oocytes after IVM (P < 0.001 and P < 0.05, respectively). Confocal microscopic observations revealed a significant increase of Cdk4 protein expression in the cytoplasm of oocytes during the maturation process. The localisation of Cx43 changed from zona pellucida before to cytoplasm of oocytes after IVM. It is supposed that the increased expression of Cdk4 and Cx43 mRNA in oocytes after IVM is linked with the accumulation of a large amount of templates during the process of oocyte maturation. The translocation especially of Cx43 from the zona pellucida into the cytoplasm may be associated with a decrease in gap junction activity in fully grown porcine oocytes. Both Cdk4 and Cx43 can be used as checkpoints of oocyte maturation.
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