Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0021
R. Knox, J. Taibl, M. Altmyer, S. Breen, D. Canaday, A. Visconti
Follicle selection and maturation for determining ovulation rate in the pig appears to occur during much of the follicular phase and up to the time of ovulation. There is evidence to suggest that counts of follicles classified as ovulatory-sized at onset of estrus may not reflect the final corpora lutea counts. Follicle size heterogeneity has been reported at estrus and may be related to increased embryonic asynchrony and mortality (Hunter et al. 1989). It is not dear which follicles ovulate at estrus but reports indicate follicles > 4 mm are LH responsive (Dufour & Mariana 1993; Lucy et al. 2001). Counts and size measures for ovulatory follicles may differ by the follicle size classification system and in response to whether follicles were assessed by physical or ultrasound measurement (Soede et al. 1998; Knox et al. 2002; Bracken et al. 2006). We hypothesized that the numbers of follicles classified as ovulatory at estrus may not reflect expected ovulation rate or litter size. We performed two experiments to characterize the changes in follicle populations from onset of estrus (Experiment 1) and from time of weaning to ovulation (Experiment 2). In experiment 1, our objectives were to measure proportions of weaned sows having large, medium and small follicles on the two days before ovulation. A total of 21 sows that had expressed estrus and ovulated between day 2 and 3 and which had real-time ultrasound digital video recordings for both the left and right ovaries on the first (period 1) and second day (period 2) of estrus were included in this study. The images of the ovaries were obtained transrectally using an Aloka 500V ultrasound with a 7.5 MHz linear transducer. The images were digitally recorded and follicles individually counted and measured using a digital display system that was calibrated to the measures of the ultrasound. The follicles were classified as small (S, <3.5 mm), Medium 1 (M1, 3.5-4.99 mm), Medium 2 (M2, 5.0-6.49 mm), Large 1 (L1, 6.5-7.99 mm), Large 2 (L2, 8.0-9.49 mm), and Large 3 (L3, 9.5-12.0 mm). Data were analyzed using the GLM procedures of SAS for the main effects of sow and period (day 1 and 2 of estrus). The response measures included the proportions of sows having the specified size class, numbers of follicles in class, and the size of the follicles. Period did not affect the percentage (22%), number (<3 follicles), or size of small follicles (3.1 mm). Period during estrus also did not affect the percentage of sows with Mt follicles (90%), or their size (4.4 mm), but numbers were reduced (P< 0.05) in period 2 (6.5 vs. 4.5). Period did not affect the percentage of sows with (100%) or numbers (14.1 follicles) of M2, but size was increased (P< 0.05) in period 2 (5.8 vs. 5.9 mm). Period did not affect the percentage of sows having L1 (100%), but numbers (7.8 vs. 9.1, P—0.01) and size (7.2 vs. 7.3, P<0.05) were both increased in period 2. Period tended (P<0.10) to increase the percentage of sows having L2 in period 2
{"title":"Assessment of follicle population changes in sows from day of weaning and during estrus using real-time ultrasound.","authors":"R. Knox, J. Taibl, M. Altmyer, S. Breen, D. Canaday, A. Visconti","doi":"10.1530/biosciprocs.18.0021","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0021","url":null,"abstract":"Follicle selection and maturation for determining ovulation rate in the pig appears to occur during much of the follicular phase and up to the time of ovulation. There is evidence to suggest that counts of follicles classified as ovulatory-sized at onset of estrus may not reflect the final corpora lutea counts. Follicle size heterogeneity has been reported at estrus and may be related to increased embryonic asynchrony and mortality (Hunter et al. 1989). It is not dear which follicles ovulate at estrus but reports indicate follicles > 4 mm are LH responsive (Dufour & Mariana 1993; Lucy et al. 2001). Counts and size measures for ovulatory follicles may differ by the follicle size classification system and in response to whether follicles were assessed by physical or ultrasound measurement (Soede et al. 1998; Knox et al. 2002; Bracken et al. 2006). We hypothesized that the numbers of follicles classified as ovulatory at estrus may not reflect expected ovulation rate or litter size. We performed two experiments to characterize the changes in follicle populations from onset of estrus (Experiment 1) and from time of weaning to ovulation (Experiment 2). In experiment 1, our objectives were to measure proportions of weaned sows having large, medium and small follicles on the two days before ovulation. A total of 21 sows that had expressed estrus and ovulated between day 2 and 3 and which had real-time ultrasound digital video recordings for both the left and right ovaries on the first (period 1) and second day (period 2) of estrus were included in this study. The images of the ovaries were obtained transrectally using an Aloka 500V ultrasound with a 7.5 MHz linear transducer. The images were digitally recorded and follicles individually counted and measured using a digital display system that was calibrated to the measures of the ultrasound. The follicles were classified as small (S, <3.5 mm), Medium 1 (M1, 3.5-4.99 mm), Medium 2 (M2, 5.0-6.49 mm), Large 1 (L1, 6.5-7.99 mm), Large 2 (L2, 8.0-9.49 mm), and Large 3 (L3, 9.5-12.0 mm). Data were analyzed using the GLM procedures of SAS for the main effects of sow and period (day 1 and 2 of estrus). The response measures included the proportions of sows having the specified size class, numbers of follicles in class, and the size of the follicles. Period did not affect the percentage (22%), number (<3 follicles), or size of small follicles (3.1 mm). Period during estrus also did not affect the percentage of sows with Mt follicles (90%), or their size (4.4 mm), but numbers were reduced (P< 0.05) in period 2 (6.5 vs. 4.5). Period did not affect the percentage of sows with (100%) or numbers (14.1 follicles) of M2, but size was increased (P< 0.05) in period 2 (5.8 vs. 5.9 mm). Period did not affect the percentage of sows having L1 (100%), but numbers (7.8 vs. 9.1, P—0.01) and size (7.2 vs. 7.3, P<0.05) were both increased in period 2. Period tended (P<0.10) to increase the percentage of sows having L2 in period 2 ","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"199-200"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48524639","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0016
Hunter Mg, F. Paradis
The mechanisms controlling the follicular growth continuum in the pig involve the interaction between local growth factors which are expressed throughout development and extra-follicular factors such as gonadotrophins. A large number of follicular growth factors, many belonging to the transforming growth factor-beta (TGF-beta) superfamily, have been identified in the somatic cells and in the oocyte. The relative importance of these intra-follicular factors varies with stage of development. The initiation of follicular growth and early preantral development is controlled locally (by factors including c-kit-kit ligand, members of the bone morphogenetic family (e.g BMP-15) and growth differentiation factor-9 (GDF-9)) and gonadotrophins are not thought to be involved until later. During antral follicle development, the oocyte secretes factors that stimulate porcine granulosa cell proliferation and differentiation, modulate apoptosis and suppress progesterone production, thereby preventing premature luteinisation. Likely candidates for mediating these effects include BMP-6, -15 and GDF-9 that are critical for fertility and ovulation rate in several mammals. There are also paracrine interactions between the somatic cells, with theca derived transforming growth factor beta (TGF-beta) playing a key role in regulating antral follicle maturation. Finally, during the periovulatory period, members of the EGF family from the granulosa cells stimulate cumulus expansion and oocyte maturation. Evidence indicates that some of these local factors may also influence oocyte developmental potential, emphasizing further the complexity, and importance, of these intra-follicular interactions.
{"title":"Intra-follicular regulatory mechanisms in the porcine ovary.","authors":"Hunter Mg, F. Paradis","doi":"10.1530/biosciprocs.18.0016","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0016","url":null,"abstract":"The mechanisms controlling the follicular growth continuum in the pig involve the interaction between local growth factors which are expressed throughout development and extra-follicular factors such as gonadotrophins. A large number of follicular growth factors, many belonging to the transforming growth factor-beta (TGF-beta) superfamily, have been identified in the somatic cells and in the oocyte. The relative importance of these intra-follicular factors varies with stage of development. The initiation of follicular growth and early preantral development is controlled locally (by factors including c-kit-kit ligand, members of the bone morphogenetic family (e.g BMP-15) and growth differentiation factor-9 (GDF-9)) and gonadotrophins are not thought to be involved until later. During antral follicle development, the oocyte secretes factors that stimulate porcine granulosa cell proliferation and differentiation, modulate apoptosis and suppress progesterone production, thereby preventing premature luteinisation. Likely candidates for mediating these effects include BMP-6, -15 and GDF-9 that are critical for fertility and ovulation rate in several mammals. There are also paracrine interactions between the somatic cells, with theca derived transforming growth factor beta (TGF-beta) playing a key role in regulating antral follicle maturation. Finally, during the periovulatory period, members of the EGF family from the granulosa cells stimulate cumulus expansion and oocyte maturation. Evidence indicates that some of these local factors may also influence oocyte developmental potential, emphasizing further the complexity, and importance, of these intra-follicular interactions.","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"149-64"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47778886","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0025
B. Freking, J. Miles, S. Bischoff, S. Tsai, N. Hardison, Y. Xia, D. Nonneman, J. Vallet, J. Piedrahita
Direct single trait selection for 11 generations resulted in a 1.6 pig advantage for uterine capacity (UC) while average birth and placental weights at term remained unchanged. Uterine capacity was defined as the total number of fully-formed pigs produced to term when ovulation rate was not limiting, using a unilateral hysterectomy-ovariectomy model. A serial slaughter experiment conducted throughout gestation determined the critical time period for the line difference in litter size was already established between d 25 and 45 of gestation and generated direct evidence of differential relative growth rates for placental tissues at these times. Timing of line differences in fetal survival as well as anecdotal evidence of tissue structural differences pointed to the developing placental tissue as a target of particular interest. Our objective was to gain insight into placental transcriptional changes during this critical stage of gestation and identify genetic loci impacted by quantitative selection for uterine capacity. Thirty gilts each from the UC and control (CO) lines were subjected to unilateral hysterectomyovariectomy at approximately 160 d of age and mated within line at approximately 280 d. Gilts were slaughtered at d 25, 30, or 40 of gestation. Fetal and placental tissues were obtained from each live embryo. Fetal liver samples were used to extract DNA and determine sex of each fetus by PCR. Two male and two female embryos closest to the litter mean for placental weight were chosen to represent each litter sampled (n 3 litters per line and time point combination). Placental tissues were pooled within litter and total RNA was extracted. Samples were labeled and hybridized to Affymetrix porcine array chips (n — 18) using the manufacturers suggested protocols. Signal intensities were normalized using GC content Robust Multi-array Average (GCRMA) on the probe level data. Filtering was based on perfect match intensities as implemented for Affymetrix Human arrays. Two-way ANOVA (two lines and three stages) was performed. Threshold values were set at a minimum of 1.5 fold difference and the false discovery rate was set to P < 0.05 (Benjamini and Hochberg algorithm). Lessstringent two-way comparisons (t-tests)were also conducted between lines within each gestation stage. GeneSifter® software web tools were utilized to conduct the analyses and generate the gene lists. An additional analysis was conducted to examine the potential for a bioinformatics method of identifying single feature polymorphism (SNP) targets between the two lines associated with the behavior of the expression data. A gene by gene linear mixed model of probe intensity variation from 11 probes per gene target was investigated to identify line x probe interactions. Log2-transformed perfect-match intensities for all observations were fit to a linear mixed model that broadly corrected for effects of breed and array. A gene-specific mixed model was fit to the normalized intensities
直接单性状选择11代后，在平均出生体重和足月胎盘重量保持不变的情况下，子宫容量(UC)优势为1.6。采用单侧子宫-卵巢切除模型，将子宫容量定义为在排卵率不受限制的情况下足月生产的完全成形猪的总数。在整个妊娠期间进行的一系列屠宰实验确定了胎仔数差异的关键时期已经确定在妊娠第25天至第45天之间，并产生了胎盘组织在这些时期相对生长速度差异的直接证据。胎儿存活的时间线差异以及组织结构差异的轶事证据表明，发育中的胎盘组织是一个特别感兴趣的目标。我们的目的是深入了解在这个关键妊娠阶段胎盘转录的变化，并确定受子宫容量定量选择影响的基因位点。分别来自UC系和对照系(CO)的30头后备母猪在大约160天龄时进行单侧子宫卵巢切除术，并在大约280天的时候进行配种。后备母猪在妊娠第25、30或40天被屠宰。从每个活胚胎中获得胎儿和胎盘组织。取胎儿肝脏标本提取DNA，用PCR法测定胎儿性别。选择最接近胎盘重量平均值的两个雄性和两个雌性胚胎来代表每胎(每条线和时间点组合n 3胎)。将胎盘组织放入凋落物中，提取总RNA。将样品标记并杂交到Affymetrix猪阵列芯片(n - 18)上，使用制造商建议的方案。利用GC含量鲁棒多阵列平均(GCRMA)对探测级数据进行信号强度归一化处理。过滤是基于完美匹配强度为Affymetrix人类阵列实现。进行双向方差分析(两条线，三个阶段)。阈值设置为至少1.5倍的差异，假发现率设置为P < 0.05 (Benjamini和Hochberg算法)。在每个妊娠阶段的品系之间也进行了较不严格的双向比较(t检验)。使用GeneSifter®软件web工具进行分析并生成基因列表。研究人员还进行了一项额外的分析，以检验生物信息学方法在与表达数据行为相关的两条细胞系之间识别单特征多态性(SNP)靶点的潜力。利用基因线性混合模型研究了每个基因靶标11个探针的探针强度变化，以确定线与探针的相互作用。所有观测值的log2转换的完美匹配强度拟合到一个线性混合模型，该模型广泛地校正了品种和阵列的影响。基因特异性混合模型拟合归一化强度(第一个模型的残差)，考虑了固定品种、探针和不同探针之间的相互作用效应，以及随机阵列效应。产生统计证据的探针(qvalue = 0.05以解释多次测试和fold change I a 2)被确定为含有SNP的候选探针。声明了交互作用
{"title":"Impact of selection for uterine capacity on the placental transcriptome.","authors":"B. Freking, J. Miles, S. Bischoff, S. Tsai, N. Hardison, Y. Xia, D. Nonneman, J. Vallet, J. Piedrahita","doi":"10.1530/biosciprocs.18.0025","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0025","url":null,"abstract":"Direct single trait selection for 11 generations resulted in a 1.6 pig advantage for uterine capacity (UC) while average birth and placental weights at term remained unchanged. Uterine capacity was defined as the total number of fully-formed pigs produced to term when ovulation rate was not limiting, using a unilateral hysterectomy-ovariectomy model. A serial slaughter experiment conducted throughout gestation determined the critical time period for the line difference in litter size was already established between d 25 and 45 of gestation and generated direct evidence of differential relative growth rates for placental tissues at these times. Timing of line differences in fetal survival as well as anecdotal evidence of tissue structural differences pointed to the developing placental tissue as a target of particular interest. Our objective was to gain insight into placental transcriptional changes during this critical stage of gestation and identify genetic loci impacted by quantitative selection for uterine capacity. Thirty gilts each from the UC and control (CO) lines were subjected to unilateral hysterectomyovariectomy at approximately 160 d of age and mated within line at approximately 280 d. Gilts were slaughtered at d 25, 30, or 40 of gestation. Fetal and placental tissues were obtained from each live embryo. Fetal liver samples were used to extract DNA and determine sex of each fetus by PCR. Two male and two female embryos closest to the litter mean for placental weight were chosen to represent each litter sampled (n 3 litters per line and time point combination). Placental tissues were pooled within litter and total RNA was extracted. Samples were labeled and hybridized to Affymetrix porcine array chips (n — 18) using the manufacturers suggested protocols. Signal intensities were normalized using GC content Robust Multi-array Average (GCRMA) on the probe level data. Filtering was based on perfect match intensities as implemented for Affymetrix Human arrays. Two-way ANOVA (two lines and three stages) was performed. Threshold values were set at a minimum of 1.5 fold difference and the false discovery rate was set to P < 0.05 (Benjamini and Hochberg algorithm). Lessstringent two-way comparisons (t-tests)were also conducted between lines within each gestation stage. GeneSifter® software web tools were utilized to conduct the analyses and generate the gene lists. An additional analysis was conducted to examine the potential for a bioinformatics method of identifying single feature polymorphism (SNP) targets between the two lines associated with the behavior of the expression data. A gene by gene linear mixed model of probe intensity variation from 11 probes per gene target was investigated to identify line x probe interactions. Log2-transformed perfect-match intensities for all observations were fit to a linear mixed model that broadly corrected for effects of breed and array. A gene-specific mixed model was fit to the normalized intensities ","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"207-8"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47940316","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0002
Pei-Shiue Tsai, B. Gadella
Fertilization is a decisive moment in life and enables the combination of the DNA from two gametes to ultimately form a new organism. The sperm surface, especially the head area, has distinguishable subdomains that are involved in distinct fertilization processes. It is known that the sperm head surface undergoes constant remodelling during epididymal maturation and migration in the male and female genital tract. But intriguingly, the identity, origin and spatial ordering of proteins at the sperm surface that are involved in mammalian fertilization are essentially unknown. This review deals with sperm surface protein modifications that are under somatic cell control. As soon as the sperm is released from the seminiferous tubules it is subjected to these modifications. These surface reorganisations continue until the sperm reside in the fallopian tube where they meet the oocyte and may fertilize it. Most likely, a selective process allows only functionally mature and intact sperm to optimally interact and fertilize the oocyte. Recent data suggest that even the perivitelline fluid is involved in sperm surface remodelling as it contains factors which could facilitate the first penetrating sperm to fertilize the oocyte. In this contribution, the kinetics of proteins at the sperm surface will be overviewed. Better understanding of this would help to design strategies to improve male fertility or to devise novel contraceptives.
{"title":"Molecular kinetics of proteins at the surface of porcine sperm before and during fertilization.","authors":"Pei-Shiue Tsai, B. Gadella","doi":"10.1530/biosciprocs.18.0002","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0002","url":null,"abstract":"Fertilization is a decisive moment in life and enables the combination of the DNA from two gametes to ultimately form a new organism. The sperm surface, especially the head area, has distinguishable subdomains that are involved in distinct fertilization processes. It is known that the sperm head surface undergoes constant remodelling during epididymal maturation and migration in the male and female genital tract. But intriguingly, the identity, origin and spatial ordering of proteins at the sperm surface that are involved in mammalian fertilization are essentially unknown. This review deals with sperm surface protein modifications that are under somatic cell control. As soon as the sperm is released from the seminiferous tubules it is subjected to these modifications. These surface reorganisations continue until the sperm reside in the fallopian tube where they meet the oocyte and may fertilize it. Most likely, a selective process allows only functionally mature and intact sperm to optimally interact and fertilize the oocyte. Recent data suggest that even the perivitelline fluid is involved in sperm surface remodelling as it contains factors which could facilitate the first penetrating sperm to fertilize the oocyte. In this contribution, the kinetics of proteins at the sperm surface will be overviewed. Better understanding of this would help to design strategies to improve male fertility or to devise novel contraceptives.","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"23-36"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43639212","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0024
J. Bijttebier, K. Tilleman, D. Deforce, M. Dhaenens, A. Van Soom, D. Maes
Follicular fluid, as a transudate of serum, constitutes the micro environment of the maturing oocyte. In a previous study, we have shown that follicular fluid (pFF) is superior to autologous serum in promoting cumulus expansion during in vitro maturation (IVM) of porcine oocytes (Bijttebier et al. 2008). After ultrafiltration of both fluids, the fraction containing molecules > 30kDa includes the factor(s) responsible for the observed differences in cumulus expansion Bifftebier, unpublished observations). This suggests the factor is likely to be a protein. The present study aimed to identify those proteins responsible for the observed differences in cumulus expansion after IVM in 10% serum ( > 30 kDa) versus 100/opFF (>30 kDa) obtained from sows in the preovulatory stage of the estrous cycle. Shotgun proteomics analysis of the pFF and serum fractions > 30kDa from 3 sows was performed by application of the 'isobaric Tag for Relative and Absolute Quantitation'(iTRAQ) technology (Applied Biosystems) followed by 2D-LC ESI-Q-TOF MSMS. Of each sample, 100pg protein material was loaded and runs were performed in duplicate. The processed data, obtained from Mascot Daemon, was searched against the pig ESTdatabase for protein identification (http:// pigest.ku.dk). Protein ratios resulting from duplicate runs were averaged, log-transformed and analyzed by Student's t-test. In addition, 600 prepubertal gilt oocytes were matured in vitro for 26h in NCSU23 supplemented with 100/0pFF ( > 30kDa) or 10% serum (> 30kDa) of each of the 3 sows. After IVM, the expanded cumulus matrices were collected and subjected to proteomic analysis. Proteins in the matrix extracts were separated using 2D-PAGE. Two spots that were absent in matrices matured in pFF were excised and submitted to mass spectrometric analysis using ESI-Q-TOF MSMS. The processed data, obtained from Mascot Daemon, was searched against the pig ESTdatabase for protein identification. First of all, serum and pFF were not depleted for high abundant proteins like albumin, because the depleted sample did not show the same biological effect on the IVM of porcine oocytes. Therefore an exclusion list was used based on the first run to exclude abundant peptides derived from albumin. Proteomic analysis of serum and pFF revealed 63 unique proteins present in both fluids of which 13 showed significantly (P< 0.05) different expression levels (10 proteins levels on P<0.01). Seven of these proteins were more abundant in serum whereas 6 of them were more abundant in the pFF fractions. Alpha2-macroglobulin (A2M) and ch4 and secrete domains of swine IgM, which were both down regulated in pFF, were also identified as the protein spots that were absent in the cumulus matrices after IVM in 10% pFF ( > 30 kDa) compared to IVM in 10% serum ( > 30 kDa). In conclusion, 2 proteins that are upregulated in autologous serum were also solely retrieved in the cumulus matrices of oocytes matured in 10% serum ( > 30 kDa). One of them, A2M
卵泡液作为血清渗出物，构成成熟卵母细胞的微环境。在之前的一项研究中，我们发现卵泡液(pFF)在促进猪卵母细胞体外成熟(IVM)过程中的积云扩张方面优于自体血清(Bijttebier et al. 2008)。在对两种流体进行超滤后，含有分子bbbb30kda的部分包括导致观测到的积云膨胀差异的因素(Bifftebier，未发表的观测结果)。这表明这个因素很可能是一种蛋白质。本研究旨在确定那些蛋白质，这些蛋白质负责观察到10%血清(>30 kDa)和100/opFF (>30 kDa)在排卵周期前获得的母猪IVM后积云扩张的差异。采用应用Applied Biosystems公司的“isobaric Tag for Relative and Absolute Quantitation”(iTRAQ)技术，采用2D-LC ESI-Q-TOF MSMS对3头母猪的pFF和血清组分> 30kDa进行了散弹枪蛋白质组学分析。每个样品装入100pg蛋白材料，重复运行。处理后的数据来自Mascot Daemon，在猪est数据库(http://www.pigest.ku.dk)中进行蛋白质鉴定。重复运行产生的蛋白质比率取平均值，进行对数变换，并通过学生t检验进行分析。另外，在NCSU23中分别添加100/0pFF (> 30kDa)或10%血清(> 30kDa)，将600个青春期前后备金卵母细胞体外成熟26h。IVM后，收集扩展的积云基质并进行蛋白质组学分析。利用2D-PAGE分离基质提取物中的蛋白质。将pFF中成熟的基质中缺失的两个斑点切除，并使用ESI-Q-TOF MSMS进行质谱分析。处理后的数据来自Mascot Daemon，与猪est数据库进行蛋白质鉴定。首先，血清和pFF没有被白蛋白等高富集蛋白所耗尽，因为耗尽的样品对猪卵母细胞的IVM没有相同的生物学效应。因此，在第一次运行的基础上使用排除列表来排除来自白蛋白的丰富肽。血清和pFF的蛋白质组学分析显示，两种液体中均存在63种独特蛋白，其中13种蛋白的表达水平与10%血清(bbb30 kDa)中的IVM有显著(P< 0.05)差异(10种蛋白的表达水平在p30 kDa)。综上所述，在10%血清(bbb30 kDa)成熟的卵母细胞积云基质中也能单独检索到2种在自体血清中上调的蛋白。其中一个，A2M，
{"title":"Proteomic study to identify factors in follicular fluid and/or serum involved in in vitro cumulus expansion of porcine oocytes.","authors":"J. Bijttebier, K. Tilleman, D. Deforce, M. Dhaenens, A. Van Soom, D. Maes","doi":"10.1530/biosciprocs.18.0024","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0024","url":null,"abstract":"Follicular fluid, as a transudate of serum, constitutes the micro environment of the maturing oocyte. In a previous study, we have shown that follicular fluid (pFF) is superior to autologous serum in promoting cumulus expansion during in vitro maturation (IVM) of porcine oocytes (Bijttebier et al. 2008). After ultrafiltration of both fluids, the fraction containing molecules > 30kDa includes the factor(s) responsible for the observed differences in cumulus expansion Bifftebier, unpublished observations). This suggests the factor is likely to be a protein. The present study aimed to identify those proteins responsible for the observed differences in cumulus expansion after IVM in 10% serum ( > 30 kDa) versus 100/opFF (>30 kDa) obtained from sows in the preovulatory stage of the estrous cycle. Shotgun proteomics analysis of the pFF and serum fractions > 30kDa from 3 sows was performed by application of the 'isobaric Tag for Relative and Absolute Quantitation'(iTRAQ) technology (Applied Biosystems) followed by 2D-LC ESI-Q-TOF MSMS. Of each sample, 100pg protein material was loaded and runs were performed in duplicate. The processed data, obtained from Mascot Daemon, was searched against the pig ESTdatabase for protein identification (http:// pigest.ku.dk). Protein ratios resulting from duplicate runs were averaged, log-transformed and analyzed by Student's t-test. In addition, 600 prepubertal gilt oocytes were matured in vitro for 26h in NCSU23 supplemented with 100/0pFF ( > 30kDa) or 10% serum (> 30kDa) of each of the 3 sows. After IVM, the expanded cumulus matrices were collected and subjected to proteomic analysis. Proteins in the matrix extracts were separated using 2D-PAGE. Two spots that were absent in matrices matured in pFF were excised and submitted to mass spectrometric analysis using ESI-Q-TOF MSMS. The processed data, obtained from Mascot Daemon, was searched against the pig ESTdatabase for protein identification. First of all, serum and pFF were not depleted for high abundant proteins like albumin, because the depleted sample did not show the same biological effect on the IVM of porcine oocytes. Therefore an exclusion list was used based on the first run to exclude abundant peptides derived from albumin. Proteomic analysis of serum and pFF revealed 63 unique proteins present in both fluids of which 13 showed significantly (P< 0.05) different expression levels (10 proteins levels on P<0.01). Seven of these proteins were more abundant in serum whereas 6 of them were more abundant in the pFF fractions. Alpha2-macroglobulin (A2M) and ch4 and secrete domains of swine IgM, which were both down regulated in pFF, were also identified as the protein spots that were absent in the cumulus matrices after IVM in 10% pFF ( > 30 kDa) compared to IVM in 10% serum ( > 30 kDa). In conclusion, 2 proteins that are upregulated in autologous serum were also solely retrieved in the cumulus matrices of oocytes matured in 10% serum ( > 30 kDa). One of them, A2M","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"205-6"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47207530","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0037
G. Johnson, F. Bazer, R. Burghardt, T. Spencer, Guoyao Wu, K. Bayless
This review highlights information on conceptus-uterus interactions in the pig with respect to uterine gene expression in response to estrogens and interferons (IFNs) secreted from elongating conceptuses. Pig conceptuses release estrogens for pregnancy recognition, but also secrete IFNs that do not appear to be antiluteolytic. Estrogens and IFNs induce expression of largely non-overlapping sets of genes, and evidence suggests that pig conceptuses orchestrate essential events of early pregnancy including pregnancy recognition signaling, implantation and secretion of histotroph by precisely controlling temporal and spatial (cell-specific) changes in uterine gene expression through initial secretion of estrogens, followed by cytokines including IFNG and IFND. By Day 12 of pregnancy, estrogens increase the expression of multiple genes in the uterine luminal epithelium including SPP1, STC1, IRF2 and STAT1 that likely have roles for implantation. By Day 15 of pregnancy, IFNs upregulate a large array of IFN responsive genes in the underlying stroma and glandular epithelium including ISG15, IRF1, STAT1, SLAs and B2M that likely have roles in uterine remodeling to support placentation.
{"title":"Conceptus-uterus interactions in pigs: endometrial gene expression in response to estrogens and interferons from conceptuses.","authors":"G. Johnson, F. Bazer, R. Burghardt, T. Spencer, Guoyao Wu, K. Bayless","doi":"10.1530/biosciprocs.18.0037","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0037","url":null,"abstract":"This review highlights information on conceptus-uterus interactions in the pig with respect to uterine gene expression in response to estrogens and interferons (IFNs) secreted from elongating conceptuses. Pig conceptuses release estrogens for pregnancy recognition, but also secrete IFNs that do not appear to be antiluteolytic. Estrogens and IFNs induce expression of largely non-overlapping sets of genes, and evidence suggests that pig conceptuses orchestrate essential events of early pregnancy including pregnancy recognition signaling, implantation and secretion of histotroph by precisely controlling temporal and spatial (cell-specific) changes in uterine gene expression through initial secretion of estrogens, followed by cytokines including IFNG and IFND. By Day 12 of pregnancy, estrogens increase the expression of multiple genes in the uterine luminal epithelium including SPP1, STC1, IRF2 and STAT1 that likely have roles for implantation. By Day 15 of pregnancy, IFNs upregulate a large array of IFN responsive genes in the underlying stroma and glandular epithelium including ISG15, IRF1, STAT1, SLAs and B2M that likely have roles in uterine remodeling to support placentation.","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"321-32"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41762607","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0023
Frankie J. White, E. M. Kimball, G. Wyman, D. Stein, Jason W. Ross, Ashworth, R. Geisert
Embryonic loss during early gestation limits litter size in swine production. Failure of the conceptus to attach properly to the uterine surface may contribute to the high rate of embryonic loss observed in swine. Attachment to the uterine surface is a highly synchronized event that requires precise communication between the expanding conceptus and endometrial tissue. Conceptus attachment to the uterine surface includes upregulation of adhesion molecules at the maternal/fetal interface for attachment as well as a pregnancy specific inflammatory response. Trophinin and osteopontin are cell adhesion molecules that may function in initial attachment between conceptus trophectoderm and uterine luminal epithelium of the pig and human. Leukocytes infiltrate the endometrium during implantation, and the pro-inflammatory cytokines Cyclooxygenase (COX)-1 and COX-2 are expressed in human and pig endometrium during pregnancy, where they are proposed to regulate conceptus implantation and uterine angiogenesis. Interleukin-10 (IL-113)increases during implantation in the mouse, human and pig and may regulate uterine inflammatory cytokines. Estrogen also controls uterine events necessary for attachment and implantation of the mouse and pig conceptus and may act in synergy with IL-1f3 to prepare the uterus for the implanting embryo. Furthermore, trophinin expression was induced by IL-1I3 in human endometrial cells, and uterine osteopontin expression is regulated by estrogen in the pig and mouse. The objective of the current study was to evaluate the hypotheses that estrogen regulates the uterine inflammatory response induced by IL-113during the establishment of pregnancy. Cyclic gilts were treated with corn oil or estradiol cypionate (5 mg) on Day 11 of the estrous cycle. On Day 12, gilts were subjected to mid-ventral laparotomy and uterine horns were randomly infused with either saline or porcine IL-113(15 pg). Uterine horns were removed at 4h and 36h post-infusion (4 gilts/trt/sampling periods) and endometrial mRNA was quantified by quantitative RT-PCR. Estrogen did not influence (P> 0.1) concentrations of endometrial COX-1 and COX-2 mRNA; however, IL-ui increased (P — 0.01) endometrial COX-2 mRNA by 3.5 fold and tended (P = 0.06) to increase COX-1 mRNA by 2.5 fold 4h post infusion. Cyclooxygenase-1 and COX-2 regulate uterine prostaglandin secretion, which is essential to normal implantation and pregnancy in pigs (Kraeling et al. 1985). Cyclooxygenase-2 null mice are infertile and fail to implant; however, implantation is not impeded in the Cox-1 null mouse (Lim et al. 1997). Although the conceptus induces uterine COX-2 expression at implantation sites, estrogen did not increase COX-2 mRNA in ovariectomized mice which is true in our pig study (Chakraborty et al. 1996). Furthermore, IL-11)regulates ovulation in mice through COX-2 and prostaglandin production (Davis et al. 1999). We hypothesize that conceptus IL-1p regulates uterine prostaglandin secretion by inc
妊娠早期胚胎丢失限制了猪产仔数。胚胎不能正确地附着在子宫表面可能导致猪胚胎的高损失率。子宫表面的附着是一个高度同步的事件，需要在扩张的子宫和子宫内膜组织之间进行精确的沟通。Conceptus对子宫表面的附着包括母体/胎儿界面粘附分子的上调，以及妊娠特异性炎症反应。滋养素和骨桥蛋白是细胞粘附分子，可能在猪和人的母体滋养外胚层和子宫腔上皮之间的初始附着中起作用。着床过程中白细胞浸润子宫内膜，促炎性细胞因子环氧化酶(COX)-1和COX-2在妊娠期间在人和猪子宫内膜中表达，它们被认为调节着床和子宫血管生成。白细胞介素-10 (IL-113)在小鼠、人和猪的着床过程中升高，并可能调节子宫炎症细胞因子。雌激素还控制小鼠和猪胚胎附着和着床所需的子宫事件，并可能与IL-1f3协同作用，为着床胚胎准备子宫。此外，IL-1I3可诱导人子宫内膜细胞中营养素的表达，雌激素可调节猪和小鼠子宫骨桥蛋白的表达。本研究的目的是评估雌激素在妊娠建立期间调节il -113诱导的子宫炎症反应的假设。在发情周期第11天用玉米油或雌二醇(5 mg)处理循环后备母猪。第12天，后备母猪进行腹中剖腹手术，子宫角随机注入生理盐水或猪IL-113(15 pg)。分别于输注后4h和36h(4只母猪/trt/采样周期)切除子宫角，采用定量RT-PCR法定量子宫内膜mRNA。雌激素对子宫内膜COX-1和COX-2 mRNA浓度无影响(P < 0.01);IL-ui可使子宫内膜COX-2 mRNA升高3.5倍(P - 0.01)，使COX-1 mRNA升高2.5倍(P = 0.06)。环氧化酶-1和COX-2调节子宫前列腺素的分泌，对猪的正常着床和妊娠至关重要(Kraeling et al. 1985)。环氧化酶-2缺失小鼠不育，植入失败;然而，在Cox-1缺失小鼠中，植入不会受到阻碍(Lim et al. 1997)。虽然受孕诱导着床部位的子宫COX-2表达，但在去卵巢小鼠中，雌激素并没有增加COX-2 mRNA，这在我们的猪研究中是正确的(Chakraborty et al. 1996)。此外，IL-11)通过COX-2和前列腺素的产生调节小鼠的排卵(Davis et al. 1999)。我们假设IL-1p通过增加子宫内膜COX-2调节子宫前列腺素分泌。前列腺素调节血管生成
{"title":"Estrogen and interleukin-1beta regulation of trophinin, osteopontin, cyclooxygenase-1, cyclooxygenase-2, and interleukin-1beta system in the porcine uterus.","authors":"Frankie J. White, E. M. Kimball, G. Wyman, D. Stein, Jason W. Ross, Ashworth, R. Geisert","doi":"10.1530/biosciprocs.18.0023","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0023","url":null,"abstract":"Embryonic loss during early gestation limits litter size in swine production. Failure of the conceptus to attach properly to the uterine surface may contribute to the high rate of embryonic loss observed in swine. Attachment to the uterine surface is a highly synchronized event that requires precise communication between the expanding conceptus and endometrial tissue. Conceptus attachment to the uterine surface includes upregulation of adhesion molecules at the maternal/fetal interface for attachment as well as a pregnancy specific inflammatory response. Trophinin and osteopontin are cell adhesion molecules that may function in initial attachment between conceptus trophectoderm and uterine luminal epithelium of the pig and human. Leukocytes infiltrate the endometrium during implantation, and the pro-inflammatory cytokines Cyclooxygenase (COX)-1 and COX-2 are expressed in human and pig endometrium during pregnancy, where they are proposed to regulate conceptus implantation and uterine angiogenesis. Interleukin-10 (IL-113)increases during implantation in the mouse, human and pig and may regulate uterine inflammatory cytokines. Estrogen also controls uterine events necessary for attachment and implantation of the mouse and pig conceptus and may act in synergy with IL-1f3 to prepare the uterus for the implanting embryo. Furthermore, trophinin expression was induced by IL-1I3 in human endometrial cells, and uterine osteopontin expression is regulated by estrogen in the pig and mouse. The objective of the current study was to evaluate the hypotheses that estrogen regulates the uterine inflammatory response induced by IL-113during the establishment of pregnancy. Cyclic gilts were treated with corn oil or estradiol cypionate (5 mg) on Day 11 of the estrous cycle. On Day 12, gilts were subjected to mid-ventral laparotomy and uterine horns were randomly infused with either saline or porcine IL-113(15 pg). Uterine horns were removed at 4h and 36h post-infusion (4 gilts/trt/sampling periods) and endometrial mRNA was quantified by quantitative RT-PCR. Estrogen did not influence (P> 0.1) concentrations of endometrial COX-1 and COX-2 mRNA; however, IL-ui increased (P — 0.01) endometrial COX-2 mRNA by 3.5 fold and tended (P = 0.06) to increase COX-1 mRNA by 2.5 fold 4h post infusion. Cyclooxygenase-1 and COX-2 regulate uterine prostaglandin secretion, which is essential to normal implantation and pregnancy in pigs (Kraeling et al. 1985). Cyclooxygenase-2 null mice are infertile and fail to implant; however, implantation is not impeded in the Cox-1 null mouse (Lim et al. 1997). Although the conceptus induces uterine COX-2 expression at implantation sites, estrogen did not increase COX-2 mRNA in ovariectomized mice which is true in our pig study (Chakraborty et al. 1996). Furthermore, IL-11)regulates ovulation in mice through COX-2 and prostaglandin production (Davis et al. 1999). We hypothesize that conceptus IL-1p regulates uterine prostaglandin secretion by inc","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"203-4"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45784313","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0028
G. Foxcroft, W. Dixon, M. Dyck, S. Novak, J. Harding, F. Almeida
Studies of low birth weight offspring have a long history in pig science. These pigs have reduced growth potential and poor carcass quality compared to their higher birth weight littermates. In contemporary commercial sows with between 10 and 15 total pigs born/litter, between-litter differences in average birth weight appear to make the largest contribution to variation in postnatal growth performance, independent of numbers born. Low birth weight is a characteristic of a subpopulation of these sows, likely as a consequence of an imbalance between ovulation rate and uterine capacity due to ongoing selection for litter size. Based on experimental studies, we hypothesize that increased crowding at day 30 of gestation primarily affects placental development and persistent negative impacts on placental growth then affect fetal development. However, embryonic myogenic gene expression is already affected at day 30. Latent effects of metabolic state on oocyte quality and early embryonic development have also been reported. In contrast to effects of uterine crowding, the embryo is primarily affected by previous catabolism. The large body of literature on gene imprinting, and the interactions between metabolism, nutrition, and methylation state, suggest that classic imprinting mechanisms may be involved. However, the potential use of genomics, epigenomics, nutrigenomics, and proteomics to investigate these mechanisms brings new demands on experimental design and data management that present a considerable challenge to the effectiveness of future research on prenatal programming in the pig.
{"title":"Prenatal programming of postnatal development in the pig.","authors":"G. Foxcroft, W. Dixon, M. Dyck, S. Novak, J. Harding, F. Almeida","doi":"10.1530/biosciprocs.18.0028","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0028","url":null,"abstract":"Studies of low birth weight offspring have a long history in pig science. These pigs have reduced growth potential and poor carcass quality compared to their higher birth weight littermates. In contemporary commercial sows with between 10 and 15 total pigs born/litter, between-litter differences in average birth weight appear to make the largest contribution to variation in postnatal growth performance, independent of numbers born. Low birth weight is a characteristic of a subpopulation of these sows, likely as a consequence of an imbalance between ovulation rate and uterine capacity due to ongoing selection for litter size. Based on experimental studies, we hypothesize that increased crowding at day 30 of gestation primarily affects placental development and persistent negative impacts on placental growth then affect fetal development. However, embryonic myogenic gene expression is already affected at day 30. Latent effects of metabolic state on oocyte quality and early embryonic development have also been reported. In contrast to effects of uterine crowding, the embryo is primarily affected by previous catabolism. The large body of literature on gene imprinting, and the interactions between metabolism, nutrition, and methylation state, suggest that classic imprinting mechanisms may be involved. However, the potential use of genomics, epigenomics, nutrigenomics, and proteomics to investigate these mechanisms brings new demands on experimental design and data management that present a considerable challenge to the effectiveness of future research on prenatal programming in the pig.","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"213-31"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"67228189","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0035
L. J. Zak, J. Patterson, J. Hancock, D. Rogan, G. Foxcroft
{"title":"Benefits of synchronizing ovulation with porcine luteinizing hormone (pLH) in a fixed time insemination protocol in weaned multiparous sows","authors":"L. J. Zak, J. Patterson, J. Hancock, D. Rogan, G. Foxcroft","doi":"10.1530/biosciprocs.18.0035","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0035","url":null,"abstract":"","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"67228850","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0013
F. Paradis, H. Moore, S. Novak, M. Dyck, W. Dixon, G. Foxcroft
Until recently, the traditional view was that the oocyte played a passive role in folliculogenesis, relying on paracrine signalling from surrounding somatic cells to acquire developmental competence. However, recent evidence suggests that the oocyte plays an active role in the process of folliculogenesis by secreting soluble factors that act on the neighboring somatic cells. Studies using rodent and ruminant animal models have shown the importance of oocytesecreted factors for cell proliferation and differentiation, steroidogenesis, metabolism, and, in certain species, in determining ovulation rate. However, little information is available in the pig species. Therefore, the objective of the current study was to determine the effects of native oocyte-secreted factors on cumulus cell protein expression in the pig follicle. Three groups of four sows were euthanized on day 19 ± 1 after the 1° post-weaning oestrus at a time point when the pre-ovulatory follicle population was established and the oocytes should have acquired full developmental competence. Follicle size and follicular fluid oestradiol concentration were used to confirm that oocytes were derived from highly oestrogenic follicles that had not been exposed to the preovulatory-LH surge. Cumulus-oocyte complexes (n 19 ± 2 per sow) were aspirated from these large pre-ovulatory follicles, washed three times in PVA TL-HEPES, and denuded (DO) from their cumulus cells. Concomitantly, gilt ovaries were obtained from a local slaughterhouse and oocytectomized cumulus complexes (00X) were prepared by microsurgically removing the oocytes from their surrounding cumulus cells. Groups of 1600X were then cultured for 22h with or without the DOs obtained from individual sows (n = 4 groups of 00X per treatment per replicate) in 36 pl droplets of modified M199 medium under mineral oil at 38.5°C in a humidified atmosphere of 5% CO2. For each of the three replicate cultures, 3 groups of 16 00X incubated with or without DOs were pooled and subjected to 2-dimensional gel electrophoresis on 7cm IPGstrips pH 3-10 in the first dimension and 100/0SDS-PAGE slab gels in the second dimension. The SYPRO Ruby (BioRad, CA) stained gels were imaged on a Typhoon Trio (GE Healthcare). Due to incomplete isoelectric focusing for one replicate, only the gel images from the remaining two replicates were finally analysed using Progenesis SameSpots software (Nonlinear Dynamics, UK). Individual spot intensities were normalized with the total spot volume from the gel of origin. A preparative gel containing the protein from the cumulus cells of 500 cumulus-oocyte complexes was also run under the same conditions but using an 18 cm IPGstrip pH 3-10. The preparative gel was matched with the analytical gels and protein spots of interest were manually excised and sent to a mass spectrometry facility for identification by LC MS/MS (Centre Genomique du Quebec, Sainte-Foy, Canada). To confirm proper matching between the analytical and prepar
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