Characterization of agouti-signaling protein during oocyte maturation and early embryonic development to improve in vitro embryo production in cattle

Progress report for GNE21-252

Project Type: Graduate Student
Funds awarded in 2021: $14,926.00
Projected End Date: 08/31/2023
Grant Recipient: West Virginia University
Region: Northeast
State: West Virginia
Graduate Student:
Faculty Advisor:
Jianbo Yao
West Virginia University
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Project Information

Project Objectives:
  1. Determine the expression profile and spatial distribution of bovine ASIP mRNA and protein in bovine ovarian tissue, GV and MII oocytes, follicular cells, and early embryos
  2. Characterize the effect of exogenous ASIP supplementation on oocyte maturation and early embryogenesis in cattle
  3. Investigate the possible role(s) of bovine oocyte derived ASIP in cumulus cell expansion, oocyte maturation, and early embryonic development to determine if ASIP is an indicator of oocyte competence

     The purpose of this project is to elucidate the role of ASIP in folliculogenesis, oocyte maturation, and early embryonic development in cattle. The leading contributing factor to the annual $1.2 billion loss experienced by the beef cow/calf industry is early embryonic loss [1]. The utilization of reproductive biotechnologies and specifically, in vitro fertilization (IVF), over the past few decades have enabled the beef and dairy industries to substantially improve the genetics and productivity of cattle. Natural breeding only allows a cow to usually produce a single calf per year and approximately 10 calves throughout her lifetime. The use of IVF and embryo transfer can vastly increase the number of offspring a genetically valuable cow can produce throughout her lifetime. In 2017, it was reported that, for the first time in history, a larger number of bovine embryos were produced in vitro and transferred worldwide rather than in vivo which further emphasizes the importance of IVF to the beef and dairy industries [2]. However, the rate of transferable embryos produced from cumulus-oocyte complexes (COC) in vitro ranges from 30­–40% [3]. Embryos produced in vitro result in fewer pregnancies than embryos flushed from donor cows and transferred to recipients [4].  The average number of oocytes collected per oocyte pick up (OPU) for Bos taurus beef cattle is on average, 21 oocytes per session and 19 for dairy cattle [5]. Of the oocytes collected during OPU, approximately 4.25 develop into quality embryos which can be transferred into a recipient [5].

     A limiting factor of the success of bovine IVF is the knowledge of mRNAs and proteins imperative to oocyte competence and early embryonic development. The quality of the oocyte limits its ability to resume meiosis, cleave following fertilization, promote embryonic development and establishment of the pregnancy, and result in a full-term, healthy pregnancy, therefore, limiting reproductive success. The identification of mRNAs and proteins driving oocyte maturation and early embryonic development is crucial to improving the efficacy of bovine IVF technologies and reproductive success. The characterization of the transcriptome and proteome of oviductal fluid will enable better determination of the needs of the oocyte and embryo during fertilization and early embryonic development. Supplementation of culture media with the proteins found in the oviductal fluid may lead to the development of enhanced culture media with higher blastocyst rates. Analysis of RNA-Sequencing (RNA-Seq) data of bovine germinal vesicle (GV) stage oocytes obtained by our laboratory revealed the ASIP mRNA transcript was over 1000-fold more abundant in the oocyte than in other tissues including the brain, heart, lung, liver, spleen, colon, and kidney. Further investigation of ASIP may unveil a critical role of ASIP during oocyte maturation and early embryonic development in cattle. Data obtained from this research could lead to the better optimization of culture media used in the IVF process to increase the number of transferable embryos obtained by better imitating the in vivo follicle and oviduct conditions.


Materials and methods:
  1. Determine the expression profile and spatial distribution of bovine ASIP mRNA and protein in bovine ovarian tissue, GV and MII oocytes, follicular cells, and early embryo.

     To characterize ASIP expression in follicular cells including cumulus, granulosa, and theca cells, and both GV and MII oocytes, quantitative PCR (RT-qPCR) was conducted. The effect of follicle size on ASIP expression was also examined using cells and oocytes collected from small (3-5 mm) and large (8-18 mm) antral follicles. Ovaries were obtained from a local abattoir and small (n = 6) and large follicles (n = 6) were dissected for granulosa and theca cell isolation. Cumulus-oocyte complexes (COCs) were aspirated from small and large follicles and GV oocytes (pools of 10) were denuded and collected for RNA isolation. Additional COCs from small and large follicles underwent in vitro maturation (IVM) for 22-24 h to obtain MII oocytes (pools of 10) and expanded cumulus cells. RNA was isolated from all cells and oocytes, reverse transcribed into cDNA, and examined for ASIP expression via RT-qPCR. Conditions for RT-qPCR reactions will follow methods as previously published [18]. ASIP expression was normalized to the expression of RPL19 and relative expression values were calculated using the standard curve method. Differences in ASIP expression were determined by two-way ANOVA using JMP statistical software testing for the effect of follicle size and cell type. Individual mean comparisons were performed using Tukey’s HSD. Embryos were produced in vitro in our laboratory. Embryo samples collected include a pool of 20 embryos per early embryonic stage ranging from 2-cell to the blastocyst stage. Embryo expression data is currently being collected. 

     To determine the protein distribution of ASIP in bovine ovarian tissue, GV and MII oocytes, follicular cells, and early embryos, western blot analysis will be used. The ASIP antibody will be purchased. Western blot analysis will be conducted as previously published [18]. Briefly, protein will be isolated from samples and protein lysate will be separated on a 4–20% gradient ready gel. Electrophoresis will be run in 1 X Tris/Glycine/SDS running buffer for 2 h. Protein will then be transferred onto an Immun-Blot PVDF membrane in 1 X transfer buffer for 1 h. Following transfer and necessary blocking, the membrane will be incubated with the ASIP primary antibody solution overnight at 4 °C. Following washes, immunoreactive proteins will be visualized via a chemiluminescent horseradish peroxidase detection system. Results from the Western blot analysis will indicate the cell/tissue types and embryonic stages in which ASIP protein is present.   

     Immunohistochemistry will be performed to localize bovine ASIP protein in tissues and COCs (GV and MII) that exhibited ASIP protein during the previous Western blot analysis experiment. The IHC protocols that will be used have been previously established and utilized by myself and lab mates. The following IHC steps will be modified when appropriate as previously described to accommodate the staining of whole-mount COCs [19]. Matured oocytes used for this experiment will be produced via in vitro maturation (IVM) in the WVU IVF laboratory. Ovarian tissue will be collected from slaughterhouse animals and placed in 4% buffered formalin phosphate overnight. The tissue will then be embedded in paraffin and sectioned (4 mm). The sections will be mounted on microscope slides are deparaffinized using xylene, and rehydrated through a series of EtOH washes, and boiled for 6 min in 0.01M sodium citrate buffer as an epitope retrieval technique. Slides will be allowed to cool for 45 min on ice and then will be washed twice for 5 min in 1 X PBS. The tissue on slides will then be enclosed using a wax pen. The slides will then be incubated in 0.3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity. Slides will then be washed in 1 X PBS for 5 min and then blocking serum added to each slide for 20 min to block non-specific binding. Slides are then rinsed with 1 X PBS and then the Streptavidin/Biotin Blocking Kit was used to block endogenous streptavidin and biotin. Following a rinse with 1 X PBS, slides will be incubated with a blocking buffer for 30 min. The ASIP antibody will be diluted in 1 X PBS and 500 mL of the solution will then be added to the slides. An isotype control will be included to ensure any observed staining is not due to unspecific binding and added to control slides at the same concentration as the primary antibody. The primary antibody and antibody control will be left on slides overnight at 4°C. The following morning, a biotinylated secondary antibody will be added to the slides. Following a 30 min incubation with the secondary antibody solution, slides will be rinsed with 1 X PBS for 15 min. Slides will be then covered with Vectastain Elite ABC reagent for 30 min and once again, rinsed with 1 X PBS for 15 min. ImmPACT DAB peroxidase substrate will be added to tissue sections and allowed to incubate for 2–5 min. Sections will be rinsed in running tap water for 5 min followed by 9 seconds in hematoxylin. Sections are immediately rinsed with double distilled water for 2 min. Slides are then dipped in Scott’s tap water and placed in double-distilled water. Finally, slides will be dehydrated in graded alcohol followed by xylene. Coverslips will be mounted over tissues using Permount and images will be taken. The IHC data will be descriptive and will reveal the localization of ASIP indicated by the previous Western blot analysis. The localization of the protein will aid in determining the function of ASIP during follicular development, cumulus cell expansion, and oocyte maturation in cattle.


  1. Characterize the effect of exogenous ASIP supplementation on oocyte maturation and early embryogenesis in cattle


     To determine the role of ASIP on oocyte maturation and early embryonic development in cattle, recombinant bovine ASIP (rbASIP) protein was created in our laboratory by using the methodology described by previous laboratory members [18, 20]. Briefly, the expression of rbASIP will be achieved through the amplification of the open reading frame (ORF) of bovine ASIP through RT-PCR using gene-specific primers that include restriction sites for specific restriction enzymes. The resulting PCR products will then be cloned into the pGEM-T Easy TA cloning vector. Subsequent cloning in frame with the glutathione S-transferase (GST) coding sequence will occur in a second bacterial vector. Fusion proteins tagged with GST will then be produced in Escherichia coli. Following bacterial culture, ASIP protein will be eluted from bacteria and collected for the supplementation of culture medium.

     Embryos will be produced in our laboratory from oocytes collected from slaughterhouse ovaries. Oocytes will be aspirated from follicles and cultured using IVM medium supplemented with either 0, 1, 10, or 100 ng/ml of rbASIP (30 COCs per treatment; n = 3 replicates), cultured for 24 h in IVM medium, and then fertilized using frozen spermatozoa from a bull of proven fertility. Following 12 h after fertilization, additional presumptive zygotes not previously exposed to rbASIP will be cultured using in vitro culture (IVC) medium supplemented with either 0, 1, 10, or 100 ng/ml of rbASIP (30 zygotes per treatment; n = 3 replicates). The culture medium we utilize does not require a change throughout IVC. At 24–48 h post-insemination, cleavage rates of embryos will be determined. All embryos will be cultured for 7 days and the percentage of embryos that develop to the 8- to 16-cell stage (71 h post-insemination) and the blastocyst stage (7 d post-insemination) will be recorded. Data will be analyzed using ANOVA and the effect of rbASIP supplementation will be tested. Percentage data will undergo an arc-sin transformation. Differences in means will be determined using Tukey-Kramer. Statistical significance will be designated as P < 0.05.


  1. Investigate the possible role(s) of bovine oocyte derived ASIP in cumulus cell expansion, oocyte maturation, and early embryonic development to determine if ASIP is an indicator of oocyte competence


     The role of bovine ASIP will be investigated in cumulus cell expansion, oocyte maturation, and early embryogenesis through the microinjection of ASIP small interfering RNA (siRNA). Microinjection of siRNA targeting bovine ASIP mRNA will interfere with the translation of ASIP protein and lead to ASIP mRNA degradation. This will allow us to study to impact of the knock-down of endogenous ASIP on cumulus cell expansion, oocyte maturation, and early embryonic development. Procedures of the siRNA experiment will follow procedures previously published [18, 21]. Three specific siRNAs will be designed to target bovine ASIP in different regions using the custom dicer-substrate siRNA (DsiRNA) design tool (Integrated DNA Technologies) and will be generated commercially.

     Bovine embryos will be generated via IVP using slaughterhouse ovaries. Oocytes at the GV stage and presumptive zygotes 16–18 h post-insemination will be used for microinjection of ASIP siRNA. Noninjected oocytes/embryos and oocytes/embryos injected with a negative control siRNA will be included. The efficacy of ASIP siRNA in reducing protein in embryos will be analyzed by RT-qPCR and Western blot analysis. Maturation of oocytes and the development of all embryos will be recorded and reported as the percentage of oocytes that undergo cumulus cell expansion and nuclear maturation and the percentage of embryos that cleave (48 h post-insemination), develop to the 8- to 16-cell stage (71 h post-insemination), and reach the blastocyst stage (7 d post-insemination). Embryos microinjected with the ASIP siRNA that proves to be the most efficient at the ablation of mRNA and protein will be supplemented with media containing rbASIP protein produced during the previous experiment listed in objective 2. Supplementation will occur for 72 h. This will determine if the replacement of ASIP protein will rescue the development of the embryo. All treatments will contain 20-30 and will be replicated 3 times (n = 3). Statistical analysis of data will include an arcsine transformation of percentage data. Treatment differences will be analyzed by using a one-way ANOVA with the differences in embryonic development will be compared via Tukey-Kramer. Statistical significance will be designated as P < 0.05. Results from the microinjection of the study will determine whether the lack of ASIP protein in bovine oocytes impacts maturation and/or early embryonic development.


Research results and discussion:

Detection of ASIP in the bovine oocyte

A total of 85 million raw reads were generated from the sequencing of a bovine oocyte library. After quality control, 78 million clean reads were obtained. All clean reads were further mapped to the bovine genome (UMD3.1) using TopHat2[62]. The transcriptome was reconstructed using ab initio assembly software Scripture [63] and Cufflinks [64]. Transcripts reconstructed by these two assemblers were merged into a combined set of transcripts, resulting in the assembly of a total number of 42,396 transcripts from 37,678 genomic loci. All assembled transcripts were categorized using the bovine genome annotation obtained from UCSC and Ensembl genome browser. Approximately 40% of the transcripts correspond to already annotated transcripts.

The expression of the annotated transcripts in oocyte vs other bovine tissues was compared using RNA-Seq data sets of 9 bovine tissues downloaded from the NCBI SRA database (Accession number SRR594491- SRR594499). Expression of ASIP was found to be much higher in the oocyte in comparison to the other tissues (oocyte FPKM: 811.53; other tissues <0.6 FPKM; Figure 1A). Based on FPKM values, the level of ASIP expression appears to be similar to the expression levels of several known oocyte-specific genes (Figure 1B) including KPNA7 [8].

Characterization of ASIP expression in the ovarian follicle and early embryos

To characterize ASIP expression within the ovarian follicle, cumulus, granulosa, and theca cell samples were collected and transcript abundance was analyzed via RT-qPCR. Results indicate low cumulus cell expression. 

granulosa and theca cells isolated from antral follicles (Figure 2A; n = 12-16). To characterize ASIP expression throughout early embryonic development, pools of 20 oocytes (GV and MII) and embryos ranging from the 2-cell stage to the blastocyst stage of early embryonic development were collected. Mature oocytes and embryo samples were generated via IVM and IVP, respectively. Data validated the RNA-Seq results as the GV and MII oocyte was found to express ASIP. During early embryonic development, ASIP transcript abundance remains stable throughout the early cell divisions while increasing following EGA (Figure 2B; n = 4 pools of 20), and blastocysts displayed very high levels of ASIP transcript.


The effect of recombinant ASIP supplementation during IVM

As ASIP was detected in the bovine oocyte, surrounding follicular cells, and the early embryo, the effect of recombinant human ASIP (rhASIP) supplementation during IVM on the rate of early embryonic development was examined. rhASIP was utilized as it is commercially available.  rhASIP protein was supplemented to cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) at the concentrations of 0, 1, 10, 100, 500, or 1000 ng/ml (10-30 COCs/well, n = 4 replicates/treatment). The control (0 ng/ml rhASIP) blastocyst rate was 29% ± 1.8. Data from this experiment the optimal concentration of rhASIP for blastocyst development lies between 10 and 100 ng/ml as determined by the peak in Figure 3A. The addition of rhASIP to the IVM medium displayed embryotropic effects at the concentrations of 10 and 100 ng/ml as an increase in day 7 blastocyst rates (45% ± 4.2 at 10 ng/ml and 45% ± 5.5 at 100 ng/ml) was observed (Figure 3B). The 100 ng/ml treatment led to a significantly higher blastocyst rate in comparison to the control (P = 0.0113; Figure 3B) as revealed by Dunnett’s test comparing both treatment groups to the control. Meanwhile, the addition of rhASIP at the concentrations of 500 and 100 ng/ml proved to be detrimental to preimplantation embryonic development as blastocyst rates were 5% ± 2.4 and 20% ± 10, respectively.

The effect of ASIP siRNA-mediated knockdown on early embryonic development

            Expression of ASIP was observed throughout early embryonic development, therefore, we wanted to address the effects of ASIP knockdown on the rate of blastocyst development. Presumptive zygotes (n = 30-37 zygotes/treatment) were collected 12-16 h post-fertilization and injected with approximately 20 pl of either ASIP siRNA (25 µM), negative siRNA (25 µM), or remained as uninjected controls. The experiment was repeated 4 times. On day 7, blastocyst rates were examined. There was no difference in blastocyst rates between the uninjected (45% ± 2.98) and negative siRNA injected (45% ± 3.8) controls. Statistical analysis using a contrast revealed blastocyst development was significantly decreased by 13% in embryos injected with ASIP siRNA (29% ± 2.98) as shown in Figure 4 (P = 0.024)

.Day 8 blastocyst development following ASIP siRNA mediated knockdown via microinjection of zygotes. Microinjection of ASIP siRNA significantly decreased the percentage of zygotes reaching the blastocyst stage of development compared to the control and negative siRNA-injected embryos (P = 0.024; n = 5 replications of 30-38 embryos/treatment). B)

Participation Summary

Education & Outreach Activities and Participation Summary

3 Webinars / talks / presentations

Participation Summary:

Education/outreach description:

SSR2021 Poster

SSR2022 Poster



     Due to the molecular nature of the proposed work, outreach to fellow researchers and embryologists will grant the most benefit as they may offer productive feedback and implement findings in their embryo production practices. This research will determine whether ASIP has the potential to enhance bovine IVF through supplementation of culture media or the use of ASIP as a marker of oocyte competence. The use of IVF can be beneficial for both large and small-scale producers as a tool to enhance their herds' genetics and decrease the generation interval. Improvement of the efficiency of the bovine IVF process will allow beef and dairy producers to spend less time and funds to obtain embryos. If more transferable embryos can be produced from a single OPU session, the producer will increase the profitability of each session.

     To ensure the sharing of the information acquired throughout this project, I plan on attending and presenting the research at several international and highly regarded scientific meetings. I presented data from this research project at the Society for the Study of Reproduction (SSR) Annual 2021 meeting in St. Louis, MO. I presented a poster presentation entitled "The Expression of Agouti-Signaling Protein During Folliculogenesis and Oocyte Maturation in Cattle". I also plan on attending the International Congress on Animal Reproduction (ICAR) in Bologna, Italy in June of 2022. The ICAR meeting will be focused on reproductive physiology and reproductive technologies with attendees from over 40 countries with attendees including academic and industry scientists and veterinarians. In the following two years, I will also be able to reach a substantial number of fellow reproductive researchers by either presenting at both or either the American Dairy Science or IETS annual meetings. At all proposed meetings, I will be able to discuss ASIP research with leading reproductive researchers and bovine IVF industry leaders from the northeast region of the United States as well as from all over the world.

     Data obtained throughout this project are expected to result in a manuscript(s) thoroughly explaining the ASIP bovine expression pattern in the oocyte, follicular cells, and early embryo, effects of ASIP on oocyte maturation, and early embryonic development and describing pathways which ASIP mediates its effects. We will aim to submit the manuscript(s) to either Biology of Reproduction or Reproduction for publication. Both journals are frequently read by bovine embryologists, researchers, and veterinarians. All animal and human reproductive researchers will benefit from the published work as biological processes are often conserved between various species.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.