紅毛猩猩與黑猩猩精子冷凍保存及精液蛋白質組比較

Abstract

歷史上第六次物種大滅絕已經在進行中，當前物種滅絕速度是正常速度的1000倍。由於黑猩猩（Pan troglodytes）和紅毛猩猩（Pongo spp.）正歷經此物種滅絕的現象，牠們的基因多樣性也正面臨相同的風險。因此，聚焦於保護和維持遺傳多樣性的保育工作是迫切需要的。然而，由於這些物種具有嚴格的社會階級制度，加上重新安置個體時的複雜性，如何在動物園內保持一個基因多樣性的圈養族群一直是一個具有高度挑戰性的難題。精子冷凍保存是一種能夠克服上述障礙的有用工具。它不僅提供了一種有效的方法來保存和運輸珍貴的遺傳物質，也有助於後續輔助生殖技術的發展和執行。然而，在冷凍保存過程中，精子細胞遭受許多損傷，特別是由氧化壓力和滲透壓所引起的各種損傷。因此，本研究的主要目的是提升紅毛猩猩精液冷凍、解凍後之品質。我們實驗室先前的研究已經建立一套能夠降低黑猩猩精液冷凍損傷的冷凍保存方案。由於黑猩猩和紅毛猩猩為同屬人科的近似物種，因此，我們假設應用於黑猩猩的冷凍保存方案將能改善紅毛猩猩整體精液冷凍、解凍後之品質。然而，將黑猩猩的冷凍保存方案應用於紅毛猩猩時，結果並不理想（總活動力26.8%）。因此，我們試著透過蛋白質體學和功能性分析來了解兩物種間潛在的差異。我們首次完整分析了黑猩猩和紅毛猩猩精子和精漿的蛋白質組成與其功能。藉由氧化壓力防禦機制的功能性分析顯示，黑猩猩精漿中的總抗氧化能力和超氧化物歧化酶活性（superoxide dismutase）明顯高於紅毛猩猩的精漿。雖然紅毛猩猩的麩胱甘肽硫轉移酶（glutathione S-transferase）活性明顯高於黑猩猩的精漿，然而而麩胱甘肽過氧化物酶（glutathione peroxidase）活性並無顯著差異。這些結果證實，黑猩猩精漿中在抵禦氧化壓力方面具有較紅毛猩猩更強的能力。 在滲透壓防禦機制的功能性分析結果顯示，紅毛猩猩的成熟精子細胞中存在與體容量調節無關的水通道蛋白（AQP5和AQP11），而黑猩猩精子細胞則表現出與體容量調節相關的AQP7。此外，藉由不同滲透壓濃度試驗，檢測精子細胞運動能力時，我們發現紅毛猩猩在低張條件下的運動能力顯著低於黑猩猩。因此我們推論紅毛猩猩不具有對抗低滲透壓所導致之損傷的有效防禦機制。總結來說，紅毛猩猩的精子與精漿較不具有抗氧化和抗滲透壓力的能力，因此，我們建議使用抗氧化劑並改善解凍過程將可提高紅毛猩猩精液冷凍、解凍後的品質

The 6th mass extinction is already happening as the current rate of species extinction is 1000 times the normal rate. Chimpanzee (Pan troglodytes) and Orangutan (Pongo spp.) are examples of this tragic phenomenon, risking the genetic diversity of the species. Therefore, conservation efforts focusing on protecting and maintaining this genetic diversity are eminently needed. However, due to the hierarchical dominant behavior of the animals and the complexity of their relocation, keeping a genetically diverse captive zoo population has proven challenging. Sperm cryopreservation is a useful tool able to overcome these obstacles. It provides an effective way to not only preserve and transport valuable genetic resources but also allow the subsequent development and execution of assisted reproductive procedures. However, during the cryopreservation process, the sperm cells suffer many damages, especially resulting from oxidative (OxS) and osmotic (OsS) stresses. Therefore, the main aim of the present research is to improve the post-thaw (P-T) quality of orangutan ejaculates. Previous research in our laboratory has developed a cryopreservation protocol capable of preventing cryo-damages in chimpanzee. As chimpanzee and orangutan are two close members of the Hominidae family, we hypothesized that applying the cryopreservation protocol used in chimpanzee would improve the overall orangutan P-T quality. Unfortunately, when applying the chimpanzee cryopreservation protocol to orangutan, the outcome was not ideal (26.8% total motility). Therefore, comprehensive proteomic and functional analyses were used to understand the potential causes behind this unsuccessful outcome. We analyzed, for the first time, the complete proteome of spermatozoa and seminal plasma in both chimpanzee and orangutan. A multitude of differences in the defenses against OxS and OsS were revealed. Furthermore, in the functional analysis of the defenses against OxS, total antioxidant capacity and superoxide dismutase were significantly higher in chimpanzee seminal plasma (SP), glutathione S-transferase activity was significantly higher in orangutan SP, and no differences were detected in glutathione peroxidase activity. Altogether those results indicate that chimpanzee present higher OxS defenses than orangutan, especially in its SP. In the functional analysis of the OsS defenses, orangutan presented aquaporins (AQP) unrelated to volume regulation in mature sperm cells (AQP5 and AQP11), whereas chimpanzee presented AQP7, which is known to be related to volume regulation. Furthermore, when motility was measured under different osmolarities, orangutan presented significantly lower motility under hypoosmotic conditions. Overall, these results lead us to believe that orangutan does not present efficient defenses against hypoosmotic shock. In conclusion, orangutan’s lesser OxS and OsS defenses propels us to recommend the use of antioxidants and modification of thawing process to improve the P-T quality.