Conditioned medium from canine amniotic membrane-derived stem cells improved dog sperm post-thaw quality-related parameters

Abstract

Mesenchymal stem cells and their derivatives are used in clinical studies for their anti-apoptotic, anti-oxidant, immunomodulatory, and regenerative properties. Their use in reproductive medicine is increasing as they have been proved to be beneficial for infertility treatment. Mesenchymal stem cells can secrete factors that influence biological processes in target tissues or cells; these factors are either directly secreted by the cells or mediated through their derivatives. Although the amniotic membrane is easy to obtain and is a good source of stem cells, clinical trials using amniotic membrane-derived mesenchymal stem cells are still uncommon, especially in the field of reproduction and artificial reproductive technologies. This study investigated the effects of conditioned medium (CM) from canine amniotic membrane-derived MSCs (cAMSCs) on canine sperm cryopreservation. For this purpose, flow cytometry analysis was performed to characterize cAMSCs. The CM prepared from cAMSCs was subjected to proteomic analysis for the identification of proteins present in the medium. Sperm samples were treated with freezing medium supplemented with 0%, 5%, 10%, and 15% of the CM, and kinetic parameters were evaluated after 4–6 h of chilling at 4 °C to select the best concentration before proceeding to cryopreservation. Quality-related parameters of frozen–thawed sperm, including motility; kinetic parameters; viability; integrity of the plasma membrane, chromatin, and acrosome; and mitochondrial activity were investigated. The results showed that 10% of the CM significantly enhanced motility, viability, mitochondrial activity, and membrane integrity (p<0.05); the analysis of chromatin and acrosome integrity, however, showed no significant differences between the treatment and control groups. Therefore, I concluded that the addition of 10% CM derived from cAMSC in the freezing medium protected canine sperm during the cryopreservation process. This research was conducted to evaluate the cyto-protective potential of cAMSC-CM on canine sperm cryopreservation, with the hypothesis that the factors contained in the CM would enhance the post-thaw quality-related parameters. The results obtained from my study show that cAMSC-CM addition in the cryopreservation medium was beneficial to canine sperm cells during the freezing-thawing process. The innocuity and practical advantages of the CM makes it an interesting candidate for more advanced research in the field of reproduction. Moreover, its beneficial effects on sperm cryopreservation gives us more perspectives and hopes for better sperm cryopreservation protocols in endangered species, especially canine endangered species such as grey wolves, African wild dogs, Island foxes and dholes.

중간엽 줄기 세포(MSC)와 그 유도체는 항 세포사멸, 항산화, 면역 조절 및 재생 특성에 대한 임상 연구에 사용된다. 또한, 불임 치료에 유익한 것이 증명되면서 생식 의학에서의 사용이 증가하고 있다. MSC는 표적 조직이나 세포에서 생물학적 과정에 영향을 미치는 요소들을 분비할 수 있는데, 이 요소들은 세포에 의해 직접 분비되거나 유도체를 통해 매개된다. 양막은 줄기 세포의 좋은 공급원이지만 임상 실험 특히, 생식 의학이나 인공 생식 기술에서 양막 유래 MSC를 사용하는 경우는 드물다. 본 연구는 개 정액 동결보존에서 개 양막 유래 중간엽 줄기 세포(cAMSCs) 유래 조정배지(CM)의 영향을 분석하였다. 먼저cAMSCs 확인을 위해 유세포 분석이 수행되었다. 그 후, 단백질 분석을 통해cAMSC로부터 제조된 CM에 존재하는 단백질을 확인하였다. 마지막으로 동결보존 전, CM의 최적 농도 설정을 위해 각각 0 %, 5 %, 10 %, 및 15 %의 CM이 함유된 동결 배지가 정자 샘플에 처리되었다. 처리된 정자 샘플들은 4 ° C에서 4 ~ 6 시간 보관된 후 동결융해 정자 품질관련 지표(운동성, 운동학적 지표, 생존력, 원형질막과 염색질 및 첨체의 완전성 및 미토콘드리아 활성) 분석에 사용되었다. 그 결과, 염색질 및 첨체의 완전성은 모든 농도의 CM처리군에서 유의미한 차이를 보이지 않았다. 그러나, 10%의 농도로 CM처리 시 정자의 운동성, 생존력, 미토콘드리아 활성 및 멤브레인 완전성이 유의미하게 향상된 것을 확인하였다 (p <0.05). 따라서, cAMSCs 유래 CM이 10% 첨가된 동결배지는 개의 정액 동결 과정에서 품질보존에 도움을 줄 수 있다.