Comparative analysis of proteomic profiles between endometrial caruncular and intercaruncular areas in ewes during the peri-implantation period

The endometrium of sheep consists of plenty of raised aglandular areas called caruncular (C), and intensely glandular intercaruncular areas (IC). In order to better understand the endometrium involved mechanisms of implantation, we used LC-MS/MS technique to profile the proteome of ovine endometrial C areas and IC areas separately during the peri-implantation period, and then compared the proteomic profiles between these two areas. We successfully detected 1740 and 1813 proteins in C areas and IC areas respectively. By comparing the proteome of these two areas, we found 170 differentially expressed proteins (DEPs) (P < 0.05), functional bioinformatics analysis showed these DEPs were mainly involved in growth and remodeling of endometrial tissue, cell adhesion and protein transport, and so on. Our study, for the first time, provided a proteomic reference for elucidating the differences between C and IC areas, as an integrated function unit respectively, during the peri-implantation period. The results could help us to better understand the implantation in the ewes. In addition, we established a relatively detailed protein database of ovine endometrium, which provide a unique reference for further studies.     

Comparative analysis of proteomic profiles between endometrial caruncular and intercaruncular areas in ewes during the peri-implantation period

The endometrium of sheep consists of plenty of raised intercaruncular areas （IC）. In order to better understand aglandular areas called caruncular （C）, and intensely glandular the endometrium involved mechanisms of implantation, we used LC-MS/MS technique to profile the proteome of ovine endometrial C areas and IC areas separately during the peri-implantation period, and then compared the proteomic profiles between these two areas. We successfully detected 1740 and 1813 proteins in C areas and IC areas respectively. By comparing the proteome of these two areas, we found 170 differentially expressed proteins （DEPs） （P 〈 0.05）, functional bioinformatics analysis showed these DEPs were mainly involved in growth and remodeling of endometrial tissue, cell adhesion and protein transport, and so on Our study, for the first time, provided a proteomic reference for elucidating the differences between C and IC areas, as an integrated function unit respectively, during the peri-implantation period. The results could help us to better understand the implantation in the ewes. In addition, we established a relatively detailed protein database of ovine endometrium, which provide a unique reference for further studies.     

Effect of excess degradable intake protein on early embryonic development, ovarian steroids, and blood urea nitrogen on days 2, 3, 4, and 5 of the estrous cycle in mature ewes

Two trials were conducted to determine whether feeding excess degradable intake protein (DIP) during a synchronized estrous cycle and the first 5 d after breeding alters early embryonic development, ovarian steroids, or BUN concentrations in ewes. Ewes were group-fed in Trial 1 (T1) and individually fed in Trial 2 (T2) either 100 (control; T1, n = 15; T2, n = 12) or 200% (high-protein; T1, n = 16; T2, n = 12) of the NRC protein recommendation for maintenance during a synchronized estrous cycle until surgery in the next cycle. Ampullae (AMP), isthmi (IST), and uterine horns (UT) of high-protein and control ewes were removed on d 2 (T1), 3 (T2), 4 (T1), or 5 (T2) after breeding. In T1, jugular blood samples were taken once daily starting on d 2 of the synchronized cycle, and in T2 on d 2, 9, 15, 16, and 17, and in both trials from estrus (d 0) to the day of surgery. Ampullae, IST, and UT flushings were examined microscopically for the presence of embryos, embryo condition, and embryo cell number. There was no trial x treatment interaction (P > or = 0.10), so data for both trials were pooled. Concentrations of BUN were higher (P < 0.05) in high-protein-fed ewes than in control ewes during the synchronized cycle and the first 5 d of the next cycle. Progesterone concentrations of the synchronized cycle did not differ (P > 0.10) between treatments. During the first 5 d of the next cycle, estradiol-17beta concentrations were lower (P = 0.06) in high-protein-fed than in control ewes. Progesterone increased (P < 0.05) to higher concentrations by d 5 in high-protein-fed ewes than in control ewes. More (P < 0.05) embryos were found in AMP of high-protein-fed ewes than in AMP of control ewes on d 4. Fewer (P = 0.05) embryos were found in UT of high-protein-fed ewes than in UT of control ewes on d 4. More embryos were found in UT of high-protein-fed ewes than in UT of control ewes on d 5. Fewer (P = 0.05) embryos were found in IST of high-protein ewes than in the IST of control ewes on d 5. Embryos of high-protein-fed ewes had more (P < 0.05) cells than embryos from control fed ewes on d 5. Feeding ewes excess DIP protein during an estrous cycle and the first 5 d after breeding initially impeded embryo transport; thereafter, embryo transport and development through the oviduct was accelerated.     

Multiple Factors Affecting Superovulation in Poll Dorset in China

To expand the breeding flock of Poll Dorset sheep in China, multiple ovulation and embryo transfer breeding program was applied to the limited number of imported Australian Poll Dorset sheep. This study investigated the effects of FSH from three different manufacturers, parity (nulliparous vs multiparous), repeated superovulation, oestrus induction, corpus luteum regression and oestrus delay on Poll Dorset superovulation. The results showed that gonadotropin FSH from Canada Folltropin‐V (Ca‐FSH) was successfully used for superovulatory treatment with 160 mg–200 mg dosage per ewe and recovered 12.91 ± 7.80 embryos. Multiparous ewes for superovulation treatment were significantly better nulliparous ewes (p < 0.05). The successive superovalution treatment reduced significantly embryo collection but did not affect transferable embryo number. Ewes with natural oestrus resulted in significantly higher number of embryos (13.83 ± 4.64) and of transferable embryos (12.00 ± 5.76) than ewes with induced oestrus (7.00 ± 4.92; 4.22 ± 3.42) and unknown oestrus cycle (5.94 ± 3.38; 3.19 ± 2.56, p < 0.05). The delayed oestrus ewes at 24 h after superovulatory treatment produced significantly fewer embryos and transferable embryos (0.92 ± 1.51 vs 0.42 ± 0.90) than those with normal oestrus (p < 0.01). Furthermore, the more transferable embryos were recovered from ewes with normal corpus luteum than those with corpus luteum regression (5.88 ± 5.09 vs 3.59 ± 4.30 and 8.83 ± 5.75 vs. 6.66 ± 5.41, p < 0.01). These results suggest that in our farm practice, a comprehensive treatment method by using the Canadian FSH (Folltropin‐V), plus choosing multiparous and natural oestrus ewes with normal corpus luteum might obtain an optimum embryo collection and embryos transfer in sheep.     

免疫荧光检测绵羊体外受精胚胎Oct4、Cdx2表达研究

Oct4基因和Cdx2基因是附植前胚胎发育的重要调控基因,并最终调控了胎儿和胎盘的发育,也在胚胎妊娠识别和附植时调控了干扰素(interferon tau,IFNT)的表达.本研究通过体外成熟、体外受精和体外培养获得了不同发育时期的绵羊胚胎,应用免疫荧光染色探讨了Oct4在早期胚胎的表达规律,结果表明:受精卵和早期卵裂...     

Comparative analysis between endometrial proteomes of pregnant and non-pregnant ewes during the peri-implantation period

Background:Early pregnancy failure has a profound impact on both human reproductive health and animal production.2/3 pregnancy failures occur during the peri-implantation period;however,the underlying mechanism(s)remains unclear.Well-organized modification of the endometrium to a receptive state is critical to establish pregnancy.Aberrant endometrial modification during implantation is thought to be largely responsible for early pregnancy loss.Result:In this study,using well-managed recipient ewes that received embryo transfer as model,we compared the endometrial proteome between pregnant and non-pregnant ewes during implantation period.After embryo transfer,recipients were assigned as pregnant or non-pregnant ewes according to the presence or absence of an elongated conceptus at Day 17 of pregnancy.By comparing the endometrial proteomic profiles between pregnant and non-pregnant ewes,we identified 94 and 257 differentially expressed proteins(DEPs) in the endometrial caruncular and intercaruncular areas,respectively.Functional analysis showed that the DEPs were mainly associated with immune response,nutrient transport and utilization,as well as proteasome-mediated proteolysis.Conclusion:These analysis imply that dysfunction of these biological processes or pathways of DEP in the endometrium is highly associated with early pregnancy loss.In addition,many proteins that are essential for the establishment of pregnancy showed dysregulation in the endometrium of non-pregnant ewes.These proteins,as potential candidates,may contribute to early pregnancy loss.     

Early Pregnancy Diagnosis by Serum Progesterone and Ultrasound in Sheep Carrying Somatic Cell Nuclear Transfer-Derived Pregnancies

Early pregnancy diagnosis and monitoring play an important role following embryo transfer in sheep. The aims of the current study were to investigate (i) the pattern of serum progesterone profiles in sheep carrying somatic cell nuclear transfer (SCNT)‐derived (clone) pregnancies, and (ii) the frequency of pregnancy loss during development following SCNT embryo transfer. Sheep SCNT embryos were made using standard nuclear transfer techniques. Day 7 embryos were surgically transferred to oestrus‐synchronized recipients (n = 27). As a control, normal fertile ewes (n = 12) were bred by natural breeding. Serum was collected from all the ewes on the day of estrus (day 0 sample), 7 days post‐estrus (day 7 sample) and 19 days post‐estrus (day 19 sample) and every 10 days thereafter until lambing or pregnancy loss occurred. Serum progesterone (P4) was assessed using enzyme immunoassay. Pregnancy was confirmed by ultrasound scanning on day 35 of pregnancy followed by subsequent scanning every 10 days. In control ewes, pregnancy rate on day 35 was 83.3% (10/12), whereas in the ewes that received SCNT embryos, it was 22.2% (6/27; p < 0.05). The day 45 pregnancy rate in the control ewes was 83.3%, whereas in the SCNT embryo recipients it was 11.0% (p < 0.05). Hormone analysis revealed that SCNT embryo recipients exhibited a significantly lower P4 profiles at different time points in pregnancy compared to controls (p < 0.05). This study highlights the use of serum progesterone in combination with ultrasound for the investigation of embryo loss and crucial times during development of normal and SCNT embryos in sheep. Further, the serum P4 levels directly reflect the degree of placental development in these two groups.     

Embryonic migration relative to maternal recognition of pregnancy in sheep

Twenty-five crossbred ewes were utilized to examine the timing of embryonic migration relative to maternal recognition of pregnancy. These ewes also were utilized to examine whether ovine embryos synthesized estradiol-17 beta in association with embryonic elongation and intrauterine migration. Embryos were flushed on d 11 through 15 from hemiovariectomized ewes. Recovery of embryos from the uterine horn contralateral to the remaining ovary indicated that migration had occurred. Ewes subsequently were returned with rams to determine their interestrous interval. Recovered embryos were classified morphologically, their length determined and individually incubated. Changes in estradiol within the medium were determined after a 6-h incubation. Embryo migration began on d 14 (P less than .05); on consecutive days from 11 through 15, 0, 0, 0, 60 and 100% of ewes examined, respectively, had an embryo in the contralateral horn. Extended estrous cycles (greater than 20 d) were observed in 0, 0, 40, 80 and 100% of ewes examined (P less than .05) following removal of embryos on d 11 through 15 of the cycle. Ovine embryos were longer (P less than .05) on d 14 (4.8 +/- 1.1 cm length, mean +/- SE) compared with d 13 (.2 +/- .1 cm) and increased further (P less than .05) on d 15 (7.8 +/- 1.1 cm). Incidence of intrauterine migration was correlated with embryonic length (r = .83; P less than .01) and estradiol synthesis (r = .77; P less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Function of berberine on porcine in vitro fertilization embryo development and differential expression analysis of microRNAs

The effect of berberine (Ber) on in vitro fertilization (IVF) embryo development in pigs and the associated differential expression of microRNAs (miRNAs) in the embryo were investigated. NCSU‐23 embryonic culture medium was used for a control group, while NCSU‐23 embryonic culture medium added with Ber was used for a Ber group. The embryo development rates in these groups were determined, and the zygotes, 4‐ and 8‐cell embryos, and blastocysts were collected for cDNA microarray analysis. The development rates of 2‐, 4‐, 8‐cell embryos and blastocysts were significantly higher in the Ber group than those in the control group (p < 0.01). The differentially expressed miRNAs in the 8‐cell versus the 4‐cell stage in control group as well as in the 8‐cell Ber group versus the 8‐cell control group overlapped, and it was found that nine miRNAs were commonly upregulated and two of them were downregulated, while there was no overlap among the other groups. The target genes of Ber‐regulated miRNAs at the 8‐cell stage were mainly associated with the molecular pathway of nucleic acid and protein synthesis. These findings suggest that Ber may regulate the expression of miRNAs at the 8‐cell stage, which is beneficial to provide material reserves for the maternal to zygote transition of porcine embryos, thereby increasing the porcine IVF embryo development rate.     

Solexa测序技术分析鸡与鹌鹑属间杂交雌性和雄性胚胎的差异microRNAs

分别从孵化3d的已鉴定性别的鸡与鹌鹑属间杂交早期雌性和雄性胚胎组织中提取小RNA,并构建各自的cDNA文库,通过Illumina／Solexa二代测序平台对两样本SmallRNA进行高通量深度测序,结合生物信息学的方法对其属间杂交早期的雌性和雄性胚胎中已知的和预测的miRNAs的类型、长度、丰度以及参与的KEGG通路等进行了初步的统计和分析。结果显示,鸡与鹌鹑属间杂交早期雌性胚胎组织的比对序列有16058009条,雄性组织有17943294条;雌性胚胎预测得到132种miRNA,雄性胚胎预测得到460种miRNA;雌性和雄性样本间差异显著的miRNA有117个（P〈0．01）;KEGG通路分析表明,这些差异miRNAs的靶基因主要参与了wnt信号通路、MAPK信号通路、TGF-beta信号通路等与胚胎生长发育相关的途径。结果表明,就已知的miRNAs而言,在鸡与鹌鹑属间杂交雌性和雄性胚胎问存在着一定的差异,这些差异的miRNAs将为后续的功能和调控机理实验研究提供依据。     

Effect of Inter‐Breed Embryo Transfer on Lamb Growing Performance and Survival

The roles of recipient and embryo genotype in determining the growth performance and survivability of offspring based on specific behavioural differences were investigated using inter‐breed embryo transfer. This study was carried out using three recipient genotypes (Awassi, Redkaraman and Tuj) and two embryo genotypes (Charollais and Romanov) to obtain the six possible combinations of ewe and lamb genotypes. Data were collected from 71 recipient ewes (10 Redkaraman with Charollais and 15 Redkaraman with Romanov embryos; 10 Tuj with Charollais and 12 Tuj with Romanov embryos, and 12 Awassi with Charollais lambs and 12 Awassi with Romanov embryos); all ewes received two frozen‐thawed embryos. Awassi ewes had a significantly longer duration of the licking/grooming event (25.5 min, p < 0.05) than Tuj ewes. Charollais lambs were significantly (p < 0.05) more likely to require birth assistance compared to Romanov lambs. Romanov lambs were significantly more (p < 0.01) active than Charollais lambs in the first 2 h after birth; ewe breed had no effect on lamb behaviour. There was no recipient breed effect on either birth or weaning weights of lambs. Charollais lambs were recorded with higher birth (5.5 ± 0.3 kg vs 3.9 ± 0.2 kg; p < 0.001) and weaning (29.4 ± 1.2 kg vs 22.4 ± 1.9 kg; p < 0.001) weights compared to Romanov lambs. At weaning Romanov lambs had significantly higher (95% vs 75%; p < 0.05) survival rates, however, this was not significantly affected by recipient breed. It was concluded that recipient breed was not an important factor in survival and weaning performance of embryo transferred lambs from a prolific breed (Romanov) while these traits recorded for lambs from meat type (Charollais) embryos were influenced by dam breed.     

Comparative analysis between endometrial proteomes of pregnant and non-pregnant ewes during the peri-implantation period

Background

Early pregnancy failure has a profound impact on both human reproductive health and animal production. 2/3 pregnancy failures occur during the peri-implantation period; however, the underlying mechanism(s) remains unclear. Well-organized modification of the endometrium to a receptive state is critical to establish pregnancy. Aberrant endometrial modification during implantation is thought to be largely responsible for early pregnancy loss.

Result

In this study, using well-managed recipient ewes that received embryo transfer as model, we compared the endometrial proteome between pregnant and non-pregnant ewes during implantation period. After embryo transfer, recipients were assigned as pregnant or non-pregnant ewes according to the presence or absence of an elongated conceptus at Day 17 of pregnancy. By comparing the endometrial proteomic profiles between pregnant and non-pregnant ewes, we identified 94 and 257 differentially expressed proteins (DEPs) in the endometrial caruncular and intercaruncular areas, respectively. Functional analysis showed that the DEPs were mainly associated with immune response, nutrient transport and utilization, as well as proteasome-mediated proteolysis.

Conclusion

These analysis imply that dysfunction of these biological processes or pathways of DEP in the endometrium is highly associated with early pregnancy loss. In addition, many proteins that are essential for the establishment of pregnancy showed dysregulation in the endometrium of non-pregnant ewes. These proteins, as potential candidates, may contribute to early pregnancy loss.

Electronic supplementary material

The online version of this article (doi:10.1186/s40104-015-0017-0) contains supplementary material, which is available to authorized users.     

胚胎移植试验羊群发情规律的研究

为了有效提高绵羊体细胞克隆和转基因等研究工作的效率,缩短胚胎移植试验羊群中空怀个体的利用周期间隔,试验对采用不同移植方法的胚胎移植受体及供体绵羊进行了移植前后的发情规律研究。结果表明:采用自制孕激素海绵栓和进口阴道孕激素释放装置(CIDR)对绵羊进行同期发情处理时发情率均接近90%,且92%以上的个体发情起始时间均集中在撤栓后24～48 h。在胚胎移植试验结束后,未参加胚胎移植个体和子宫角法未移植受体第2情期发情起始时间主要集中在上次发情后的14～20 d,而输卵管法未移植受体和输卵管法冲卵供体的发情时间则比较分散,呈现明显延后状态。未参加胚胎移植个体和子宫角法未移植受体人工授精后的受胎率接近往年羊场的平均水平,输卵管法未移植受体的受胎率略有下降,且输卵管法冲卵供体的受胎率明显降低。     

OBSERVATIONS ON THE BACTERIAL POPULATION OF THE OS CERVIX OF THE EWE BEFORE AND AFTER EMBRYO DEATH

Bacterial samples were isolated from the os cervix of mature Merino ewes during a normal oestrous cycle and after colchicine treatment which killed the 25-day old embryos. The population of bacteria was small during the oestrous cycle and consisted of Achromobacter spp. Alcaligenes spp, Corynebacterium spp, Bacillus spp and Escherichia coli. There was a significant increase in the numbers of bacteria isolated and a change in the proportions of the bacteria isolated from ewes after embryo death. Much greater numbers of potentially pathogenic organisms, including Pasteurella multocida, Corynebacterium pyogenes, Staphylococcus spp and Streptococcus spp were isolated after embryos were killed. It is suggested that persistence of potential pathogens in the vagina may impair fertility in ewes at the first oestrus after embryo death.     

Functional Morphology of the Zona Pellucida

The zona pellucida (ZP) is an extracellular matrix surrounding the oocyte and the early embryo that exerts several important functions during fertilization and early embryonic development. The ZP of most mammalian species is composed of three major glycoproteins that show considerable heterogeneity due to extensive post-translational modifications. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of the ZP reveals three to four glycoproteins which have been nominated ZPI. ZP2, ZP3 and ZP4. As cloning and characterization of the ZP genes of a variety of mammalian species including domestic animals show a high homology, three classes of ZP genes, ZPA, ZPB and ZPC can be discerned. The corresponding proteins were named ZPA, ZPB and ZPC. Whereas in the mouse ZPB is the primary sperm receptor. the situation is more complicated in other species. For instance, in the pig ZPA has been shown to possess receptor activity. Interaction between gametes during fertilization is at least in part regulated by carbohydrate moieties of the ZP and carbohydrate-binding proteins of the sperm surface. In domestic animals zona proteins are expressed in both the oocyte and granulosa cells in a stage-specific pattern and may play a role in granulosa cell differentiation. The role of ZP glycoproteins in immunocontraception is briefly discussed.     

Preliminary observations on reproduction in a female sheep-goat chimaera.

Four oestrous cycles of a female sheep-goat chimaera were monitored by using a vasectomised ram. The mean (+/- se) length of the cycle was 18.5 +/- 0.64 days with a range from 17 to 20 days. The chimaera was superovulated twice, bred to fertile rams, and the embryos recovered by laparotomy 13 or five days after oestrus, so that karyotype analysis could reveal the genotype of the oocyte. After the first superovulation one ovine day-13 embryo was collected; two fragments of another embryo (or embryos) were also collected, but readable chromosome spreads were not obtained from these embryos or from the two four-cell embryos that were collected five days after the second superovulation. Two surgical embryo transfers to the chimaera resulted in pregnancies. The first transfer involved an eight-cell ovine embryo and two caprine morulae and ended in the abortion of an ovine fetus between days 110 and 130. The second pregnancy occurred after the transfer of two ovine and two caprine morulae. A healthy lamb was born on day 147 of pregnancy. Both placentae had small numbers of cotyledons. A histological evaluation of the cotyledons revealed an abnormal placentome structure in the first pregnancy but not in the second.     

绵羊miRNA-1185-5p的基因组定位、靶基因预测和功能分析

miRNAs是一类小分子非编码RNAs,在机体许多生理及病理过程中发挥调控作用。该课题组前期研究发现,miRNA-1185-5p在经产双羔母羊卵巢中表达水平显著低于经产单羔母羊。为进一步深入挖掘miRNA-1185-5p在绵羊生殖过程中的作用,利用生物信息学手段分析了miRNA-1185-5p的基因组定位,预测其靶基因,并对靶基因进行GO功能分析。结果表明,miRNA-1185-5p是位于绵羊18号染色体的内含子miRNA;利用miRDB和microT 2种方法预测获得miRNA-1185-5p的23个靶基因,这些靶基因主要参与细胞增殖、分化和凋亡过程以及代谢等生物学过程,其中,靶基因AhR直接参与生殖过程。该研究说明miRNA-1185-5p可能通过作用于AhR等靶基因对绵羊生殖过程发挥调控作用。     

Tauroursodeoxycholic acid enhances the pre-implantation embryo development by reducing apoptosis in pigs

Apoptosis is an important determinant of the normal development of pre-implantation embryos in vitro. Recently, endoplasmic reticulum (ER) stress-mediated apoptosis has been extensively investigated in a wide variety of diseases. Efficient functioning of the ER is essential for most cellular activities and survival. Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, has been reported to attenuate ER stress-mediated cell death by interrupting the classic pathways of apoptosis. Therefore, in this study, the anti-apoptotic effect of TUDCA on ER stress-induced apoptosis was examined in pre-implantation pig embryos. Also, tunicamycin was used to investigate the effects of ER stress on pig embryo development. After in vitro maturation and fertilization, presumptive pig embryos were cultured in NCSU-23 medium supplemented with TUDCA or TM for 6 days at 39 °C, 5% CO(2) in air. All data were analysed using one-way anova and Duncan's multiple range test in the statistical analysis system (SAS). In addition, we also determined the optimal TM and TUDCA concentrations. Samples were treated with TM at concentrations of 0, 1, 2 or 5 μm and with TUDCA at concentrations of 0, 100, 200 or 300 μm. When TM was used during in vitro culture, only 8.2% (8/97) of the embryos developed to the blastocyst stage when the treatment concentration was 1 μm compared with 27.4% (28/102) of the embryos in the control group (p < 0.05). In contrast, the frequency of blastocyst formation and the number of cells were higher when treated with 200 μm TUDCA compared with the control group (32.8% and 39.5 vs 22.2% and 35.6, p < 0.05). Moreover, the developmental rate to the blastocyst stage embryo in the group treated with TM and TUDCA was not significantly different from that of the control group (17.8%, 26/142 vs 24.9%, 36/145). Furthermore, the blastocyst cell number was enhanced (31.9 vs 36.9) and apoptosis reduced (TUNEL-positive nuclei number, 6.0 vs 3.2) by TUDCA treatment in pig embryos. In the real-time quantitative RT-PCR analysis, the expression of anti-apoptotic Bcl-XL gene was shown to be increased in the blastocyst stage because of TUDCA treatment, whereas expression of pro-apoptotic Bax was decreased. In addition, we also found that TUDCA decreased the rate of TM-induced apoptosis in the pre-implantation stage. Taken together, our results indicate that TUDCA improves the developmental competence of pig embryos by modulating ER stress-induced apoptosis during the pre-implantation stage.