Theca cell‐conditioned medium added to in vitro maturation enhances embryo developmental competence of buffalo (<i>Bubalus bubalis</i>) oocytes after parthenogenic activation

Self Cite

Granulosa cells (GCs) play a crucial role in follicular development and atresia. Previous studies have showed that GCs in the form of monolayer influenced in vitro maturation (IVM) of oocytes. However, the effects of GCs in the form of conditioned medium and monolayer on IVM and development competence of buffalo oocytes remain unclear. In the present study, we examined the impacts of GC‐conditioned medium (GCCM) and monolayer GC on maturation efficiency and embryo development of buffalo oocytes after parthenogenetic activation (PA). Our results showed that GCCM that was collected on day 2 and added to IVM medium at a 20% proportional level (2 days and 20%) exerted significant negative effects on IVM rate (41.6% vs. 44.5%), but significantly enhanced embryo development (oocyte cleavage, 81.3% vs. 69.3%; blastocyst formation, 36.3% vs. 29.3%) of buffalo oocytes after PA compared with the control group. Furthermore, monolayer GC significantly reduced both maturation efficiency (40.2% vs. 44.5%) and embryo development (oocyte cleavage, 60.6% vs. 69.3%; blastocyst formation, 20.6% vs. 29.3%) of buffalo oocytes after PA compared to the control group. Our study indicated that GCs in the form of GCCM (2 days and 20%) and monolayer GC had different effects on IVM and subsequent parthenogenetic development of buffalo oocytes.

Section: Discussionsupporting

confidence: 93%

“…However, monolayer GC significantly reduced both maturation rate and parthenogenetic development of buffalo oocytes. Remarkedly, the effects of GCs on oocyte IVM are not entirely consistent with that of theca cells in buffalo, as has been confirmed by the previous published article (Zhang, Deng, Chen, et al., 2020).…”

Section: Discussionmentioning

confidence: 52%

Granulosa cells affect in vitro maturation and subsequent parthenogenetic development of buffalo (Bubalus bubalis) oocytes

Zhang

Wang

et al. 2021

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“…PA was performed as described in previous reports (Zhang et al., 2020). After oocyte maturation, the surrounding cumulus cells were gently removed using a pipette.…”

Section: Methodsmentioning

confidence: 99%

Follistatin (FST) is expressed in buffalo (Bubalus bubalis) ovarian follicles and promotes oocyte maturation and early embryonic development

Wang,

Chen,

Shen

et al. 2023

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Follistatin (FST), a member of the transforming growth factor‐β (TGF‐β) superfamily, has been identified as an inhibitor of follicle‐stimulating hormone. Previous studies showed that it plays an important role in animal reproduction. Therefore, this study aims to investigate its effect on the maturation of buffalo oocytes in vitro, and the underlying mechanism of FST affecting oocyte maturation was also explored in buffalo cumulus cells. Results showed that FST was enriched in the ovary and expressed at different stages of buffalo ovarian follicles as well as during oocyte maturation and early embryo development. The FST expression level was up‐regulated in MII buffalo oocytes compared with the GV stage (p < .05). To study the effects of FST on buffalo oocytes' maturation and early embryonic development, we added the pcD3.1 skeleton vector and PCD3.1‐EGFP‐FST vector into the maturation fluid of buffalo oocytes, respectively. It was demonstrated that FST promoted the in vitro maturation rate of buffalo oocytes and the blastocyst rate of embryos cultured in vitro (p < .05). By interfering with FST expression, we discovered that FST in cumulus cells plays a crucial role in oocyte maturation. Interference with the FST expression during the buffalo oocyte maturation did not affect the first polar body rate of buffalo oocyte (p > .05). In contrast, the location of mitochondria in oocytes was abnormal, and the cumulus expansion area was reduced (p < .05). After parthenogenetic activation, the cleavage and blastocyst rates of the FST‐interfered group were reduced (p < .05). Furthermore, RT‐qPCR was performed to investigate further the underlying mechanism by which FST enhances oocyte maturation. We found that overexpression of FST could up‐regulate the expression level of apoptosis suppressor gene Bcl‐2 and TGF‐β/SMAD pathway‐related genes TGF‐β, SMAD2, and SMAD3 (p < .05). In contrast, the expression levels of SMAD4 and pro‐apoptotic gene BAX were significantly decreased (p < .05). The FST gene could affect buffalo oocyte maturation by regulating the oocyte mitochondria integrity, the cumulus expansion, cumulus cell apoptosis, and the expression levels of TGF‐β/SMAD pathway‐related genes.

“…One of the most important factors affecting the SCNT efficiency are the origin and quality of nuclear donor cells (Agrawal et al, 2018; Em et al, 2016; Lu et al, 2011; Skrzyszowska et al, 2008; Wiater, Samiec, Skrzyszowska, et al, 2021). The SCNT efficiency is also affected by the synchronization of the processes of meiotic, cytoplasmic and epigenomic maturation in the in vitro cultured nuclear recipient oocytes (Gorczyca et al, 2022; Zhang et al, 2020). Additionally, pivotal factors that determine the cloning efficiency are: (1) the capability of donor cell nuclei to undergo epigenomic reprogramming of their transcriptomic signatures (Raja et al, 2019; Ruan et al, 2018; Saini & Selokar, 2018; Samiec & Skrzyszowska, 2005, 2021), and (2) the molecular interrelations between nuclear and mitochondrial genomes in SCNT‐derived embryos (Samiec, 2005; Srirattana et al, 2011; Takeda, 2013).…”

Section: Introductionmentioning

confidence: 99%

Comparison the effects of 5‐Aza‐2′‐deoxycytidine and zebularine on the in vitro development, blastocyst quality, methylation pattern and conception rate on handmade cloned buffalo embryos

Saha

Chauhan

Manik

et al. 2022

In this study we treated the handmade cloned (HMC) buffalo embryos with the DNA methylation inhibitors; 5-aza-2′-deoxycytidine (AzadC) or Zebularine individually after post-fusion and during in vitro culture till eighth day. The blastocysts production rate significantly improved (p < .01) after treating embryos independently with 5 nM AzadC and 5 nM zebularine compared with 2 and 10 nM AzadC or zebularine groups, respectively. The highest cleavage rates were obtained for 5 nM treatment of AzadC and zebularine compared with other treatments and untreated control group. Quality of blastocysts were evaluated using total cell number (TCN) and the ratio of number of inner cell mass (ICM) cells/total cell number (ICM/TCN). Zebularine treatments (2/5/10 nM) significantly improved both TCN and ICM/TCN ratio compared with AzadC treatments (2/5/10 nM); however, control group TCN and ICM/TCN ratio was found lower. The methylation percentage of pDS4.1 and B. bubalis satellite DNA were comparatively more attenuated with 5 nM zebularine than 5 nM AzadC treatment. The increased in vitro development rates of the treated embryos were correlated with the decreased level of DNA methylation and the improved blastocyst quality. Following transfer of 5 nM zebularine treated embryos to 6 recipients, 4 were found to be pregnant, though the pregnancies were not carried to full term.