Dadson, Adwoa
(2025)
The effect of whole ovary cryopreservation in sheep.
PhD thesis, University of Nottingham.
Abstract
The potential of whole ovary cryopreservation (WOCP) to restore ovarian function and
fertility has been demonstrated in sheep, resulting in multiple live births from primordial and
preantral follicles. However, the impact of WOCP on oocytes from antral follicles, their viability,
and the long-term effects on ovarian tissue, oocytes, and subsequent embryos remain unclear. This
study aimed to investigate the viability and maturation capacity of antral follicle oocytes following
WOCP, along with variations in the transcriptomic profile and DNA methylation patterns in sheep
ovarian tissue.
The study commenced with in vitro maturation (IVM) of antral follicle oocytes, assessing
nuclear maturation using DAPI after 24 hours. WOCP's immediate effects on oocyte viability and
apoptosis were examined using propidium iodide (PI) and TUNEL, respectively. Chromatin
configuration of germinal vesicle (GV) oocytes and qualitative analysis of connexin 37 and 43
were performed to assess cell-to-cell communication. A comprehensive transcriptomic analysis
using microarray and global DNA methylation assessment was performed on ovarian tissue post
WOCP. Lastly, a scoping review to examine evidence related to epigenetic changes associated to
oocyte and ovarian tissue cryopreservation (OTC).
The evaluation of nuclear maturation revealed a significantly lower maturation rate in the
WOCP group (8%, n = 1/64) compared to the control (fresh) group (67%, n = 60/90). Proportions
of PI-stained cells were relatively close between the WOCP and control groups (fresh group 17.1%;
WOCP group 38.6%, p > 0.05). However, a significant difference in apoptotic index was observed
between the control (fresh) group (26.6%) and WOCP (75.4%). Cryoprotectant exposure
predominantly induced a condensed pattern of chromatin fibres (SNE- 54%) in GV-stage oocytes,
with noticeable decondensed patterns (NSN- 36%) and abnormal condensation, highlighting
cryopreservation-induced alterations. Microarray analysis identified 2557 genes out of an initial
24,596 genes as statistically significant (p<0.05), but none remained significant after FDR
correction. Despite this, 114 genes were selected for further analysis as potential candidates for
differential expression. Gene Ontology (GO) enrichment analysis revealed changes in fundamental
cellular activities, including metabolic processes, biological regulation, and cell proliferation, with
significant positive enrichment in signalling and transport pathways, and negative enrichment in
methylation-related annotations. KEGG pathway analysis showed upregulation of ribosomal genes
(e.g., RPL11, RPL29) suggesting increased protein synthesis, and neurodegenerative disease-
associated genes (e.g., NDUFA1, NDUFS5) integral to mitochondrial oxidative phosphorylation
and thermogenesis, indicating increased ATP demands and cold-induced metabolic adaptations.
Conversely, downregulation of autophagy (ATG16L1, ATG9A) and lysine degradation pathways
(ALDH9A1, SETMAR) was observed, suggesting compromised cellular quality control.
Furthermore, pro-apoptotic genes (BAX, STC2) were upregulated, while anti-apoptotic BCL2 was
downregulated, indicating increased cellular susceptibility to apoptosis. Downregulation of
transcriptional regulators (zinc finger proteins) and key signalling pathways like WNT4, GRM7,
and GRM8 was also identified, suggesting disruptions to ovarian development and follicle
activation. Global DNA methylation exhibited no significant difference between the control group
and WOCP. The review highlighted that cryopreservation, particularly vitrification, induced
significant epigenetic alterations, including DNA methylation, histone modification, and
microRNA expression changes, in oocytes and ovarian tissue across species.
In conclusion, this study unveils the intricate molecular and cellular responses of antral
follicle oocytes to WOCP. The observed detrimental effects emphasise the challenges in
preserving delicate ovarian tissue structures, underscoring the necessity for optimized
cryopreservation protocols. The study highlights the crucial role of cumulus cells in oocyte
maturation and stresses the importance of their integrity during cryopreservation. Furthermore,
insights into chromatin configuration and DNA methylation patterns provide valuable information
on adaptive responses and epigenetic changes induced by WOCP. Dysregulation of gene
expression in WOCP ovaries, particularly in pathways associated with metabolic processes and
follicular development, underscores the complexity of reproductive outcomes post
cryopreservation. Overall, the findings underscore the ongoing need for refinement in
cryopreservation techniques to ensure the preservation of ovarian tissue integrity and optimize
reproductive outcomes.
| Item Type: |
Thesis (University of Nottingham only)
(PhD)
|
| Supervisors: |
Hernandez-Medrano, Juan
Watkins, Adam |
| Keywords: |
Ovarian tissue integrity; Antral follicle oocytes; Transcriptomic profile; DNA methylation patterns; Cryopreservation |
| Subjects: |
Q Science > QL Zoology > QL605 Chordates. Vertebrates |
| Faculties/Schools: |
UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine |
| Item ID: |
81960 |
| Depositing User: |
Dadson, Adwoa
|
| Date Deposited: |
31 Dec 2025 04:40 |
| Last Modified: |
31 Dec 2025 04:40 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/81960 |
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