Transcriptional Drug Repositioning Improves Development Of Somatic Cell Nuclear Transfer Embryos Via Inhibition Of CDK4/6 And Cyclin D1 During First Cleavage of Bovine Pseudo-Zygotes

Paria Behdarvandiyan ACECR Institute of Higher Education (Isfahan Branch) Sayedeh Sahar Hosseini ACECR Institute of Higher Education (Isfahan Branch) Farnoosh Jafarpour Royan Institute for Biotechnology, ACECR Mehdi Hajian Royan Institute for Biotechnology, ACECR Morteza Hosseini Royan Institute for Biotechnology, ACECR Mohsen Rahimi Royan Institute for Biotechnology, ACECR Reza Moradi Royan Institute for Biotechnology, ACECR Yasaman Kalantar Motamedi University of Cambridge Andreas Bender (  ab454@cam.ac.uk ) University of Cambridge Mohammad Hossein Nasr-Esfahani Royan Institute for Biotechnology, ACECR


Introduction
The somatic cell nuclear transfer (SCNT) technique involves implanting a donor nucleus from a somatic (body) cell into enucleated oocyte and it requires extensive nuclear reprogramming. This reprogramming consists of erasing the former epigenetic memory in the somatic cell nucleus and adding "de novo" epigenetic information, which leads to conferring the totipotent state to the newly forming embryos. 1 As a practical application, the rst SCNT-derived calves were generated in 1998 by Cibelli and his colleagues. 2 Although bovine species have relatively higher reproductive cloning e ciency compared to other mammals, cloning e ciency is still less than 10% [3][4][5] , which is far less than full-term developmental e ciency of in vivo and in vitro fertilized embryos 6-8 This problem is caused by incomplete nuclear reprogramming of SCNT embryos, and hence methods are currently being sought to improve this situation.
One of the most important factors responsible for the insu ciency of early and late embryonic development following SCNT is abnormal transcriptional reprogramming. [9][10][11] This phenomenon is mainly due to different biological nature of transferred somatic cells compared to specialized gametes, the sperm and the oocyte during fertilization. [12][13][14][15][16] In this regard, using various epigenetic drugs such as DNA methyltransferase inhibitors (DNMTis) 17-19 , histone deacetylase inhibitors (HDACis) [20][21][22][23][24] and histone methyltransferase inhibitors (HMTis) 25,26 have become increasingly common to improve the e ciency of transcriptional reprogramming during SCNT by modifying the epigenetic status of donor cells and/or reconstructed oocytes. 10,27 Various DNMTis and HDACis have been extensively used to improve epigenetic reprogramming in SCNT derived embryos in different species. [28][29][30][31] Several studies have shown that this approach can signi cantly increase the e ciency of early and/or full-term development in different species. [32][33][34][35][36] In one of our previous studies, we assessed the effect of assisted epigenetic modi cations in broblast donor cells using trichostatin A (TSA) on the transcriptome of bovine SCNT blastocysts with a microarray chip. In that study, it was found that TSA markedly changed epigenetic reprogramming, reconstituted oocytes (5mC, 5hmC) and blastocysts, but some canonical pathways (including WNT and FGF) were similarly affected in both control and SCNT treated groups. 37 Even though there are known small molecules which are able to improve the success rate of SCNT embryo development to some extent, there is still a need to identify new classes of small molecules with different mechanisms of action, which more e ciently improve the e ciency of SCNT in both in vitro and in vivo conditions. In practice, due to nancial and labor cost of the experiment, it is not possible to test a large number of small molecules on in vitro produced embryos. It is challenging to identify which small molecules to pick for this purpose, and this is precisely what the current work addresses: In this study, transcriptional drug repositioning has for the rst time been successfully applied for improving SCNT outcome.
Transcriptional drug repositioning has recently attracted increasing attention as it enables to study gene expression pro les of different diseases, identify key genes and search for small molecules that can target those genes effectively among large scale database of compound treatments. 38, 39 Its successful application to provide therapeutic starting points in oncology 40,41 and neurodegenerative diseases has been established before. 42 Some of the current authors have also used this approach in novel areas of improving stem cell differentiation to cardiomyocytes 43 and suggesting novel anti-malarial drugs 44 , and hence its utility has been shown across many application areas before.
In this particular work, for improving SCNT outcome, gene expression data of SCNT embryos was compared to gene expression of in vitro fertilized (IVF) embryos and genes that were differentially expressed were identi ed. Given that IVF has generally higher success rates than SCNT, this selection provided genes that are differentially expressed in SCNT embryos, and which on the one hand provide a possible explanation for lower success rates, and on the other hand potentially need to be transcriptionally adjusted to improve SCNT success rates (see Fig. 1 for a schematic representation of this process). The Library of Integrated Network-Based Cellular Signatures (LINCS) was used as a initial dataset for selecting such small molecules 45 . The LINCS database provides data for 20,413 compounds applied on 77 cancer cell lines (after certain time points with various concentrations) leading to 201,776 gene signatures of compound treatments which the algorithm was applied in order to rank-order compounds for the given purpose. The selected compounds were then experimentally evaluated further, as described in this work, to both validate the algorithm employed, as well as to provide practically useful novel small molecules to improve SCNT success rates. Results mRNA expression of CDK4 and 6 and CCND1, 2 and 3 in MII oocytes, sperm, bovine fetal broblast cells, and embryos derived from IVF and SCNT Given that Palbociclib and Fenretinide are selective chemical inhibitor of CDK4/6 and inhibitor of CCND1, rst we investigated mRNA expression levels of CDK4/6 and CCND1/2/3 in MII oocyte, sperm, broblasts, and 6 and 12 hpi/hpa IVF and SCNT embryos under untreated conditions. Total RNA was successfully isolated from MII oocytes, sperm, and bovine fetal broblast cells (BFFs) using different methods. In the rst step, the mRNA expression of CDK4/6 and CCND1/2/3 in MII oocytes, sperm and BFFs was evaluated and were normalized to the geomean of two previously validated endogenous reference genes, GAPDH and β-ACTIN (Fig. 2, A-E). While the mRNA expression of CDK4/6 was remarkably low in sperm, the expression levels were signi cantly higher in MII oocytes and BFFs (P < 0.05, Fig. 2). The mRNA expressions of CCND1/2/3 were signi cantly lower in sperm compared to BFFs but just lower compared to MII oocytes (Fig. 2). In addition, the expression of these genes was also signi cantly higher in BFFs compared to MII oocytes. Furthermore, the mRNA expression of aforementioned genes was characterized in IVF derived embryos 6 and 12 hpi and also in SCNT derived embryos 6 and 12 hpa. As shown in the result (Fig. 2), the expression of CDK4/6 and CCND1/2/3 has a similar pattern, of being signi cantly higher in SCNT derived embryos compared to IVF derived embryos.
In addition, the level of expression of these genes in SCNT embryos was similar to MII oocytes.
We can conclude that mRNA expression of CDK4/6 and CCND1 is signi cantly higher in SCNT embryos compared to IVF embryos, and hence SCNT embryos may bene t from treatment with Palbociclib and/or Fenretinide through inhibition of CDK4/6 and CCND1 and slowing down the rst cleavage.
Determination of the optimal concentration of Palbociclib and Fenretinide in parthenogenesis and parthenogenetic bovine embryos In order to obtain the optimum concentration of Palbociclib and Fenretinide, the parthenogenetically produced embryos were treated with selected concentrations of Palbociclib (50,100,200, 400 and 800 nM) and Fenretinide (4, 8 and 16 µM). These concentrations were selected because the concentrations of instances of Palbociclib and Fenretinide in the LINCS database were 400nM and 4µM, respectively (See Supplementary Table 1). Given that the G1 phase in parthenogenetic embryos is approximately 9 h, we limited the treatment duration to six hours. 46 One of the main regulators in the G 1 to S-phase transition during cell cycle is Cyclin D1 and its binding partners CDK4 and 6. 47 Since the targets of Palbociclib and Fenretinide are CDK4/6 and Cyclin D1, respectively, we next examined the two-cell formation at 30 hpa. As shown in Fig. 3A, the addition of Palbociclib at concentrations of 50, 100, 200, 400 and 800 nM did not change the proportion of embryos (P > 0.05) reaching two-cell stage at 30 hpa as compared to control embryos. In addition, no signi cant change (P > 0.05) was also observed in cleavage rate at 72 hpa (Fig. 3B). However, addition of 100 nM Palbociclib to culture medium for 6 hpa signi cantly (P < 0.05) increased the proportion of oocytes that reached the blastocyst stage at day seven after activation (Fig. 3C). In a second set of experiments, bovine activated oocytes were also treated with 0 (control), 4, 8 and 16 µM of Fenretinide for 6 hpa. As shown in Fig. 4A and 4B, addition of Fenretinide at concentrations of 4, 8, and 16 µM neither changed the two-cell formation rate nor cleavage rate at 30 and 72 hpa, respectively (P > 0.05). On the other hand, supplementation of culture medium with 4 µM Fenretinide for 6 hpa signi cantly (P < 0.05) increased the blastocyst yield at day seven compared to other treatment groups (Fig. 4C). In conclusion, while both Palbociclib and Fenretinide did not change the two-cell formation rate and cleavage rate, they showed bene cial effects on blastocyst yield in parthenogenetic embryos.
Determination of the optimal time for treatment of activated bovine oocytes with optimal Palbociclib concentrations in parthenogenetic embryos We next optimised the time of treatment of activated oocytes with the optimal concentration of Palbociclib and Fenretinide. Previously, it was shown that the observed difference between the onset of the S-phase was related to the duration of the G1-phase 46 , and in particular the S-phase started at 9.5 to 15.5 hpi or hpa in IVF or parthenogenetic embryos, respectively. Therefore, we extended the time of treatment with the optimal concentration of Palbociclib (100 nM, as obtained in the previous experiment) from 6 hpa to 9 and 12 hpa.
While extending the time of treatment of activated oocytes with 100 nM Palbociclib from 6 h to 12 h resulted in a signi cant reduction in two-cell formation rate (P < 0.05, Fig. 5A), no change was observed (P > 0.05) in cleavage rate at day three (Fig. 5B). In addition, the blastocyst rate of those activated oocytes which were treated with 100 nM Palbociclib for 6 hpa was signi cantly higher than other treatment groups (P < 0.05, Fig. 5C). Given that we did not observe any improvement in blastocyst formation rate after 12 h treatment with Palbociclib, no experiment was performed for a 15 h time point.
Optimal concentration and time of treatment of Palbociclib and Fenretinide improved blastocyst yields in parthenogenetic embryos to levels similar to those of IVF embryos Next, independent parallel groups including IVF embryos, untreated Parthenogenetic embryos, and Parthenogenetic embryos treated with either 100 nM Palbociclib for 6 hpa (PA-PALB100 6h) or with 4 µM Fenretinide for 6 hpa (PA-FEN4 6h) were carried out in triplicate (simultaneously), to assess the developmental competence of the derived embryos. As shown in Fig. 6A and 6D, the rst cleavage rate (two-cell formation rate) evaluated 30 hpa or hpi was higher in Parthenogenetic embryos compared with IVF derived embryos (P < 0.05). In addition, neither treatment with Palbociclib, nor Fenretinide, prolonged the rst cleavage (evaluated at 30 hpa) compared to the IVF group ( Fig. 6A and 6D, P > 0.05).
Subsequently, the cleavage and blastocyst rates were evaluated from the same group of oocytes/embryos. The cleavage rates assessed on day three of development were similar among the treatment groups for both Palbociclib and Fenretinide ( Fig. 6B and 6E, P > 0.05). The blastocyst rate on day seven, however showed a higher percentage in 100 nM Palbociclib for 6 hpa and 4 µM Fenretinide for 6 hpa as compared to non-treated PA group (P < 0.05) and it was similar to the IVF group ( Fig. 6C and 6F P > 0.05).

Effect of Palbociclib and Fenretinide on developmental characteristic of SCNT derived embryos
We next determined whether the optimal concentrations of Palbociclib (100 nM) and Fenretinide (4 µM) for 6 hpa (in agreement with the results in Parthenogenetic embryos) could also promote the developmental competence of SCNT derived embryos. For this purpose, we treated the SCNTreconstructed embryos with 100 nM Palbociclib for 6 hpa, and found no improvement was observed in two-cell formation (Fig. 7A), cleavage (Fig. 7B) and blastocyst ( Fig. 7C) rates (P > 0.05). This observation may be related to the different nature of SCNT embryos in comparison to Parthenogenetic embryos. In SCNT embryos a broblast cell, which has higher expression of CDK4/6 ( Fig. 2), is introduced into an enucleated oocyte, while on the other hand broblast cells are absent in Parthenogenetic embryos. So, it is conceivable that longer periods of time are needed to inhibit the function of CDK4/6 in SCNT embryos for better reprogramming. For this purpose, we extended the time of treatment post activation and treated the reconstructed embryos for 9, 12 and 15 h with the optimal concentration of Palbociclib. As shown in Fig. 7A, the two-cell formation rate was decreased signi cantly (P < 0.05) following the treatment of reconstructed embryos with 100 nM Palbociclib for 9 and 12 h in compare to other groups. In addition, the cleavage rate in the SCNT-PALB100 12h group was signi cantly higher than control group (Fig. 7B, P < 0.05). Furthermore, blastocyst formation was also signi cantly higher in the SCNT-PALB100 12h group compared to the other groups ( Fig. 7C, P < 0.05). We can hence conclude that parthenogenetic and SCNT embryos have different requirements with respect to Palbociclib treatment conditions, which we assume to be due to different expression of CDK4/6 in both cases.
The optimal concentration and time of treatment of Fenretinide in Parthenogenetic embryos were next also used in SCNT embryos. No signi cant change (P > 0.05) was observed in two-cell formation  Fig. 1C). Therefore, we conclude that parthenogenetic and SCNT embryos have different most effective treatment conditions for both Palbociclib and Fenretinide. This is likely to be due to the different nature of these two types of embryos (and associated higher expression of CDK4/6 and CCND1), which is related to the introduction of broblasts cell into enucleated oocytes in SCNT embryos, while broblast cells are absent in parthenogenetic embryos.
Optimal concentration and time of treatment of Palbociclib and Fenretinide improved developmental competence of SCNT derived embryos similar to those of IVF embryos Independent, simultaneous parallel groups including IVF embryos, untreated SCNT embryos, and SCNT embryos treated either with 100 nM Palbociclib for 12 hpa (SCNT-PALB100 12h), or treated with 16 µM Fenretinide for 6 hpa (SCNT-FEN16 6h), were carried out in triplicate to next assess the developmental competence of the derived embryos. As shown in Fig. 9A, the two-cell formation rate (which was evaluated at 30 hpa or hpi) was higher in SCNT embryos, compared with IVF-derived embryos (P < 0.05). Following treatment of SCNT-reconstructed embryos with 100 nM Palbociclib for 12 hpa the rst cleavage rate (evaluated 30 hpa) was lower compared to the SCNT group (Fig. 9A). However, despite the reduction of rst cleavage rate in the SCNT-treated group compared to the non-treated SCNT group, the rst cleavage rate remained signi cantly higher in the SCNT treated group than the IVF group (P < 0.05).
The cleavage rate assessed on day three of development was signi cantly (P < 0.05) higher in SCNT-PALB100, 12h compared to IVF and SCNT groups (Fig. 9B). The blastocyst rate on day seven showed a higher percentage in the SCNT-PALB100 12h group compared to the non-treated SCNT group, and it was in particular similar to the IVF group (P > 0.05), as shown in Fig. 9C. As shown in Fig. 9D, treating SCNT embryos with 16 µM Fenretinide for 6 hpa did not change the percentage of successful rst cleavage after 30 hpa in comparison with the non-treated SCNT group. In addition, the two-cell formation rate in the non-treated and treated SCNT groups was similar to each other, and signi cantly higher than in the IVF group. The cleavage rate at day three was signi cantly higher in the SCNT-FEN16 6h group, compared to the non-treated SCNT and IVF group (Fig. 9E). Finally, treating SCNT-reconstructed embryos with 16 µM Fenretinide for 6 hpa increased the blastocyst formation rate at day seven compared to that of the nontreated SCNT group, and to values similar to the IVF group (Fig. 9F). Overall, we can conclude that treating SCNT embryos with both Palbociclib and Fenretinide slows down the rst cleavage rate at 30 hpa. In addition, SCNT embryos bene t from treatment with Palbociclib or Fenretinide through inhibition of CDK4/6 and CCND1 in terms of cleavage and blastocyst rate, which was improved to values similar to the IVF group.
Expression of core pluripotency and trophectoderm markers in SCNT-derived blastocysts from the Palbociclib and Fenretinide treated groups We nally investigated the gene expression of two core pluripotency genes, namely POU5F1 48-50 and NANOG 51,52 , and also TEAD4 53,54 as a critical regulator of trophectoderm development, on day seven in blastocysts using real-time RT-PCR analysis. The results (Fig. 10) showed that the blastocysts derived from both the SCNT-PALB100 12h and SCNT-FEN16 6h groups express a higher level of NANOG, POU5F1 and TEAD4 mRNA compared to non-treated SCNT embryos. In addition, the mRNA expression of those genes was also signi cantly higher in SCNT-treated embryos in comparison with IVF embryos. Hence, treating the SCNT embryos with both Palbociclib and Fenretinide promote the quality of derived blastocysts in terms of expression of core pluripotency and trophectoderm markers.

Discussion
Despite many successes in the eld of SCNT and cellular reprogramming, e ciency of this technique remains a challenge for its practical application in reproduction. 55 Aberrant epigenetic reprogramming of somatic nuclei after SCNT is still considered as the main hurdle of SCNT procedure, and therefore, many studies have tried to overcome this obstacle via epigenetic modi cations (induced DNA/histone hypomethylation or histone hyper-acetylation) of somatic donor cells and/or reconstructed oocytes. 56 In this work, a novel computational approach for selecting small molecules for improving cloning e ciency has been presented. To this end, large scale gene expression pro les of compound treatments were utilized, which were then matched with differentially expressed genes between SCNT and IVFderived embryos (whose gene expression data was published earlier 37 ). Two highly ranked small molecules, Palbociclib and Fenretinide, were selected and used in this study for experimental validation.
In this study, we treated the reconstructed oocytes immediately after activation using 100 nM Palbociclib and 16 µM Fenretinide for 12 and 6 h (after optimizing conditions), respectively. Overall, we found that treating SCNT embryos in this way improved developmental competence of SCNT bovine embryos.
Palbociclib and Fenretinide are selective inhibitor of the cyclin-dependent kinases 4/6 (CDK4/6) and inhibitor of Cyclin D1, respectively (see Supplementary Tables 3 and 4 for all available pharmacology data obtainable from the ChEMBL database for the concentrations bellow what we used in this study) and are widely used for cancer therapy. 57,58 Both drugs act by inhibiting the formation of the CDK4/6-Cyclin D1 complex, and preventing the phosphorylation of Retinoblastoma protein (Rb) and thereby G1/S cell cycle transition. [59][60][61] While the oocyte is a main determinant factor of regulating cleavage, spermatozoa also plays an important role in the timing of early cleavage. 46,62,63 In this regard, zygotes derived from low fertility sperm showed a delayed rst cleavage compared with those derived from high fertility sperm. 64 To the contrary, in another study comparing high and low fertility sperm, early cleavage was more prominent in the low fertility group. 65 In addition, it has been shown before that fast-cleaved embryos, unlike slowcleaved embryos, had increased loss of methylation at H19 and Snrpn imprinted genes and aberrant H19 expression compared with in vivo counterparts. 66 This demonstrates that fast cleaved-embryos had some abnormalities in epigenetic modi cation of imprinting genes which may hamper the proper embryonic development There are various important developmental events that occur prior to the rst cleavage, including syngamy, early embryonic genome activation, and epigenetic reprogramming, which crucially affect SCNT outcomes. [67][68][69] In previous studies in bovine SCNT, it has been observed that the rst cleavage time is shortened in SCNT embryos, compared to IVF embryos. In this study, it was hypothesized that the faster cleavage in SCNT is mainly due to the de ciency of sperm-borne regulatory factors, mainly miRNAs. 70,71 Numerous studies have con rmed that sperm-borne miRNAs are important during the early embryonic development. 62,72,73 It is known that, paternal miR-34c is important for the rst cleavage 62 and sperm-borne miRNA-449b increases the time of rst cleavage and improves the epigenetic reprograming in SCNT embryos comparable to that of IVF embryos. 70 In addition, supplementation of SCNT embryos with sperm-borne small RNA prolonged the average cleavage time of SCNT embryos, which was longer than that of the control group, and similar to IVF embryos. 71 Furthermore, in this previous study the authors found that sperm-borne small RNA can affect abnormal pronuclear-like structures, and improve developmental competence of bovine SCNT embryos during both pre-and post-implantation development.
Previous studies have shown there are six miRNAs which are exclusively expressed in spermatozoa and they are absent in oocytes, 72 namely miR-34b, miR-34c, miR-99a, miR-214, miR-451, and miR-449, which commonly target genes of the CDK6 and CCND family ( Supplementary References 1-16). Thus, we hypothesized that the small molecule inhibitors of CDK4/6 and CCND1 Palbociclib and Fenretinide prolong the rst cleavage in SCNT embryos in a similar manner, and that postponing the rst cleavage in SCNT embryos acts in favor of reprogramming of broblast cells, and thereby improves SCNT e ciency.
To verify this hypothesis, rst we assessed the mRNA expression of CDK4/6 and CCND1/2/3 in MII oocyte, sperm, broblasts, and 6 and 12 hpi/hpa IVF and SCNT embryos. In this study, we observed that expression of these genes are higher in broblast than sperm. Furthermore, the relative expression of these genes are higher in SCNT embryos, compared with IVF embryos. These results are in accordance with a previous study 74 which have demonstrated a higher expression of CDK4/6 in mouse broblasts than oocytes and 6 hpi IVF mouse embryos. 74 In the current study, we treated the SCNT embryos with Palbociclib or Fenretinide post activation and investigated their effects on two cell formation after 30 hpa and embryonic development. The results show that the optimized concentration and time of treatment of both small molecules (100 nM Palbociclib for 12 hpa and 16 µM Fenretinide for 6 hpa) have an effect on reduction of two-cell formation rate, which is a consequence of prolonged rst cleavage. Mechanistically, this effect can be explained by an inhibition of the formation of the CDK4/6-Cyclin D1 complex, preventing the phosphorylation of Retinoblastoma (Rb). The consequence is the prevention of pseudozygotes from exiting the G1 phase and entering the S phase of the cell cycle and thus delayed the rst cleavage compared to that in the control SCNT group. Meanwhile, the cleavage ratio at 72 h in the experimental groups was higher than that in the control SCNT group. Furthermore, our results showed that later rst cleavage has a bene cial effect on developmental competence of SCNT embryos in terms of blastocyst yield. To further investigate the effect of Palbociclib and Fenretinide on the quality of resultant embryos, we assessed the relative mRNA expression of three developmentally important genes including NANOG, POU5F1 and TEAD4. From this we could see that while the expression of these genes in SCNT-treated blastocysts was higher than in the SCNT-control blastocyst, and also the IVF blastocyst. Whether higher mRNA expression of these genes lead to higher post-implantation development requires further investigation.
This study has hence overall demonstrated the rst application of transcriptional drug repositioning to identify two small molecules that were able to (i) prolong the time of rst cleavage in treated SCNT embryos, (ii) to increase the cleavage rate after 72 h, and (iii) bene t the development of SCNT embryo in vitro via inhibition of CDK4/6 and CCND1 during rst cleavage. This represents a novel application of transcriptional drug repurposing strategies in this eld, and it is expected that it also leads to increased SCNT e ciency in the future.

Media and reagents
All reagents and media were obtained from Sigma Chemical Co.

Somatic donor cell preparation
Bovine fetal broblast cells (BFFs) were prepared similar to the protocol which has been used in our previous studies. 75,76 Brie y, the tissue from 2-month-old female fetuses was nely minced until it became possible to pipette. Subsequently, the minced tissue was disassociated by trypsinization [0.25% trypsin/EDTA (Gibco, Invitrogen)]. Next, cell suspension was centrifuged and cultured in Dulbecco's modi ed Eagle medium F-12 (DMEM/F-12) containing 10% FBS and 1% penicillin-streptomycin at 37.5°C and 5% CO 2 in a humidi ed atmosphere. After reaching con uency, BFFs were frozen in -196 ºC liquid nitrogen. Frozen cells were thawed and used for various assessments.

Bovine oocyte preparation
The ovaries were transferred to the laboratory from slaughterhouse as soon as possible (about in 2 hours) in physiological saline, containing 100 IU/ml penicillin and 100 mg/ml streptomycin at 15-17°C. Upon arrival, the ovaries were washed, trimmed and then kept at 15-17 ° C until the time of harvesting of the oocytes according to the previously described protocol. 77 All the 2-8 mm follicles were aspirated with an 18 gauge needle attached to a vacuum pump for obtaining cumulus-oocyte complexes (COCs). Only oocytes containing homogenous cytoplasm and at least three layers of compact cumulus cells were selected for in vitro maturation (IVM).  The method which was used to produce parthenogenesis embryos was described previously. 79 In brief, matured oocytes were denuded with 300 IU/ml hyaluronidase for three minutes. Then, the denuded oocytes were activated by 5 µl Ca-inophore for ve minutes. The reconstructed oocytes were washed and transferred to 6-dimethylaminopurine (6-DMAP) which was supplemented with different concentrations of Palbociclib or Fenretinide. After 4 h incubation in 6-DMAP, the activated oocytes were washed and transferred to serum-free BO-IVF media (Cat. IVC1703N) containing the speci ed concentration of Palbociclib for 2, 5 and 8 h (6, 9 and 15 h in total or Fenretinide) or Fenretinide for 2 h (6 h in total). These time courses were selected based on a previous study which demonstrated that duration of G1 phase in parthenogenetic bovine embryos is about 9 h. 46 Six embryos were cultured in 20 µl serum-free BO-IVF media without Palbociclib or Fenretinide for seven days at 38.5 ºC, 5% CO2, 5% O2 in humidi ed air under mineral oil. Tow cell formation rate was reported 30 h after activation. In addition, cleavage and blastocyst rates were reported at day three and seven of embryonic development.

Embryo production by SCNT
The optimum concentration and duration of treatment, which were derived for Palbociclib and Fenretinide in the previous section in parthenogenetically activated embryos, were used in SCNT-derived embryos.
The method for production of SCNT embryos were largely based on those described before. 37,76 In brief, denuded oocytes were exposed to 5 mg/ml pronase for 20-30 seconds, followed by deactivation with H-TCM + 20% FBS for 15 minutes for the removal of zona pellucida. Oocyte enucleation was performed manually using a ne pulled Pasteur pipette. 76 Thereafter, zona-free oocytes were incubated in TCM supplemented with 4 µg/ml demecolcine for 1h at 38.5°C. Subsequently, the cytoplasmic protrusion, which contains MII spindle, was separated by a hand-held manual oocyte enucleation pipette. In order to transfer the somatic nucleus to the enucleated oocyte, a broblast cell was attached to the membrane of the enucleated oocyte in H-TCM medium supplemented with 10 mg/ml phytohemagglutinin. Then, the couplet of enucleated oocyte and broblast cell was electrofused using sinusoidal electric current (7 V/cm) for 10 sec, followed by two direct currents (1.75 kV/cm for 30 µsec and 1 sec delay). The procedure of activation and culture of embryos were as described in the previous section (Embryo production by parthenogenesis) for the production of parthenogenetic embryos. Tow cell formation rate was reported 30 h after activation. In addition, cleavage and blastocyst rates were reported at day three and seven of embryonic development.

Embryo production by IVF
In this study, the IVF procedure was performed as described previously 80 and used as gold standard. Frozen semen straws were thawed (30 sec in air and then 45 sec in 37°C water bath). Afterward, the semen was centrifuged at 300 g for ve minutes at room temperature (RT) to remove the cryoprotectant. The semen pellet was layered on a discontinuous Pure Sperm® gradient ( used. This technique was carried out as previously described. 76 Brie y, broblast cells, matured oocytes (MII), sperm, 6 and 12 hours after insemination of IVF embryos (5 hours were given for sperm penetration) and 6 and 12 hours after activation of SCNT embryos were used to investigate the expression level of CDK4 and 6 and CCND1, CCND2 and CCND3 genes. Expression of pluripotency (POU5F1 and NANOG) and trophectoderm (TEAD4) markers were assessed to evaluate the quality of blastocysts derived from IVF, SCNT (SCNT-CRL) and treated SCNT embryos (SCNT-TRT).       hpa. Data are expressed as the mean ± SEM. The experiments were replicated at least three times.
Different letters indicate signi cant differences among the groups (P<0.05).