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Embryo arrest and reactivation: potential candidates controlling embryonic diapause in the tammar wallaby and mink†
Jane C Fenelon1,2, Geoffrey Shaw1, Stephen R Frankenberg1
1School of BioSciences, The University of Melbourne, Victoria, Australia.
Abstract:
Embryonic diapause is a period of developmental arrest which requires coordination of a molecular cross-talk between the endometrium and blastocyst to ensure a successful reactivation, but the exact mechanisms are undefined. The objectives of this study were to screen the tammar blastocyst for potential diapause control factors and to investigate the potential for members of the epidermal growth factor (EGF) family to coordinate reactivation. A select number of factors were also examined in the mink to determine whether their expression patterns were conserved across diapause species. The full-length sequences of the tammar genes of interest were first cloned to establish their level of sequence conservation with other mammals. The uterine expression of EGF family members EGF and heparin-binding EGF (HBEGF) and their receptors (EGFR and erb-b2 receptor tyrosine kinase 4 (ERBB4)) was determined by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Both HBEGF and EGF were significantly upregulated at reactivation compared to diapause. In the blastocyst, the expression of the potential diapause factors Forkhead box class O family members (FOXO1, FOXO3, and FOXO4), tumor protein 53 (TP53), cyclin-dependent kinase inhibitor 1A (CDKN1A), and the EGF family were examined by RT-PCR and immunofluorescence. Nuclear (and hence active) FOXO expression was confirmed for the first time in a mammalian diapause blastocyst in both the tammar and the mink-CDKN1A was also expressed, but TP53 is not involved and EGFR was not detected in the blastocyst. These results indicate that the EGF family, FOXOs, and CDKN1A are promising candidates for the molecular control of embryonic diapause in mammals.
Insights
Embryonic diapause reactivation involves molecular signals. This study identified the epidermal growth factor (EGF) family, FOXO proteins, and CDKN1A as key factors in mammalian embryonic diapause.
Area of Science:
- Developmental Biology
- Reproductive Biology
- Mammalian Embryology
Background:
- Embryonic diapause is a crucial developmental arrest in mammals, requiring complex molecular communication between the uterine endometrium and the blastocyst for successful reactivation.
- The precise molecular mechanisms governing embryonic diapause and its reactivation remain largely undefined, necessitating further investigation into key regulatory factors.
Purpose of the Study:
- To screen the tammar blastocyst for potential factors controlling embryonic diapause.
- To investigate the role of epidermal growth factor (EGF) family members in coordinating blastocyst reactivation during diapause.
- To assess the conservation of these factors' expression patterns in mink, another species exhibiting diapause.
Main Methods:
- Cloning of tammar genes to determine sequence conservation across mammals.
- Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry to analyze uterine expression of EGF, heparin-binding EGF (HBEGF), and their receptors (EGFR, ERBB4).
- RT-PCR and immunofluorescence to examine blastocyst expression of Forkhead box class O (FOXO) family members (FOXO1, FOXO3, FOXO4), tumor protein 53 (TP53), cyclin-dependent kinase inhibitor 1A (CDKN1A), and EGF family members.
Main Results:
- Heparin-binding EGF (HBEGF) and EGF were significantly upregulated in the uterus during reactivation compared to the diapause state.
- Active nuclear FOXO expression was confirmed in mammalian diapause blastocysts (tammar and mink) for the first time.
- Cyclin-dependent kinase inhibitor 1A (CDKN1A) was expressed in the blastocyst, while TP53 was not involved, and EGFR was undetected.
Conclusions:
- The epidermal growth factor (EGF) family, specifically HBEGF and EGF, plays a significant role in the uterine regulation of embryonic diapause reactivation.
- FOXO proteins and CDKN1A are identified as critical intracellular factors within the blastocyst involved in controlling embryonic diapause.
- These findings highlight EGF family members, FOXOs, and CDKN1A as promising candidates for elucidating the molecular control of embryonic diapause in mammals.
