Despite advancements in assisted reproductive techniques, intracytoplasmic sperm injection (ICSI) success remains limited in ruminants, notably in sheep, suggesting underlying factors negatively affecting early embryonic development [1]. Recent investigations into Ca2+ response dynamics post-ICSI in sheep oocytes revealed aberrant patterns potentially responsible of impairing critical developmental processes beyond initial activation, and highlighted avenues for improving ICSI outcomes in these species [2]. Phospholipase C zeta (PLCζ) is a pivotal molecule in initiating oocyte activation and embryo development [3], yet its characterization in sheep remains lacking. In this study, we aimed to elucidate the localization of PLCζ in ram spermatozoa. Employing ionomycin-induced acrosome reaction and acrosome staining, we sought to investigate the correlation between the acrosome reaction and PLCζ localization, providing insight into its role in sheep reproduction. In a preliminary examination, anti-PLCζ immunocytochemistry and acrosome staining were conducted on thawed spermatozoa (CTR). Results revealed that among the analyzed spermatozoa (n=153), 13% exhibited PLCζ localization in the acrosomal region and 74% in the subequatorial position, with only 23% displaying an intact acrosome. Upon treatment with ionomycin, a progressive translocation of PLCζ was observed, aligning with the temporal and dynamic changes associated with the acrosome reaction. Within just 3 minutes of exposure to ionomycin, PLCζ exhibited a notable shift in localization, with 8% (n=174) found in the acrosomal region, and 35% in subequatorial position (P<0.05 vs CTR), accompanied by a significant reduction in intact acrosomes (14.4%, n=174, P<0.05 vs CTR). By 5 minutes of ionomycin exposure, spermatozoa demonstrated nearly complete reactivity (intact acrosome <1%, n=162), while PLCζ localization showed a further decrease in the acrosomal position (6%, P<0.05 vs CTR) and an increase in the subequatorial position (38.3%, P<0.05 vs CTR). The experimental procedures conducted are evidently preliminary, necessitating further analysis. However, the results highlight a clear relationship between the acrosome reaction and the location of PLCζ, suggesting its probable involvement in oocyte activation. Therefore, a comprehensive review of the artificial oocyte activation process would be warranted. [1] O. Briski, D.F. Salamone. Late Past, present and future of ICSI in livestock species. Anim Reprod Sci. 2022 Nov; 246:106925. [2] L. Gioia, L. Palazzese, M. Czernik, D. Iuso, H. Fulka, J. Jr. Fulka, P. Loi. Oocyte activation is a cytoplasm-confined event so far: what about the nucleus? Reproduction. 2024 Feb; 167:e230360.[3] T. Wakai, A. Mehregan, R. A. Fissore. Ca2+ Signaling and Homeostasis in Mammalian Oocytes and Eggs. Cold Spring Harb Perspect Biol. 2019 Dec; 11:a035162.
Dynamics of PLCζ During Acrosome Reaction in Frozen Ram Spermatozoa
L. Palazzese
;A. Scudieri;M. Moncada;M. Lo Sterzo;M. Czernik;L. Gioia;P. Loi
2024-01-01
Abstract
Despite advancements in assisted reproductive techniques, intracytoplasmic sperm injection (ICSI) success remains limited in ruminants, notably in sheep, suggesting underlying factors negatively affecting early embryonic development [1]. Recent investigations into Ca2+ response dynamics post-ICSI in sheep oocytes revealed aberrant patterns potentially responsible of impairing critical developmental processes beyond initial activation, and highlighted avenues for improving ICSI outcomes in these species [2]. Phospholipase C zeta (PLCζ) is a pivotal molecule in initiating oocyte activation and embryo development [3], yet its characterization in sheep remains lacking. In this study, we aimed to elucidate the localization of PLCζ in ram spermatozoa. Employing ionomycin-induced acrosome reaction and acrosome staining, we sought to investigate the correlation between the acrosome reaction and PLCζ localization, providing insight into its role in sheep reproduction. In a preliminary examination, anti-PLCζ immunocytochemistry and acrosome staining were conducted on thawed spermatozoa (CTR). Results revealed that among the analyzed spermatozoa (n=153), 13% exhibited PLCζ localization in the acrosomal region and 74% in the subequatorial position, with only 23% displaying an intact acrosome. Upon treatment with ionomycin, a progressive translocation of PLCζ was observed, aligning with the temporal and dynamic changes associated with the acrosome reaction. Within just 3 minutes of exposure to ionomycin, PLCζ exhibited a notable shift in localization, with 8% (n=174) found in the acrosomal region, and 35% in subequatorial position (P<0.05 vs CTR), accompanied by a significant reduction in intact acrosomes (14.4%, n=174, P<0.05 vs CTR). By 5 minutes of ionomycin exposure, spermatozoa demonstrated nearly complete reactivity (intact acrosome <1%, n=162), while PLCζ localization showed a further decrease in the acrosomal position (6%, P<0.05 vs CTR) and an increase in the subequatorial position (38.3%, P<0.05 vs CTR). The experimental procedures conducted are evidently preliminary, necessitating further analysis. However, the results highlight a clear relationship between the acrosome reaction and the location of PLCζ, suggesting its probable involvement in oocyte activation. Therefore, a comprehensive review of the artificial oocyte activation process would be warranted. [1] O. Briski, D.F. Salamone. Late Past, present and future of ICSI in livestock species. Anim Reprod Sci. 2022 Nov; 246:106925. [2] L. Gioia, L. Palazzese, M. Czernik, D. Iuso, H. Fulka, J. Jr. Fulka, P. Loi. Oocyte activation is a cytoplasm-confined event so far: what about the nucleus? Reproduction. 2024 Feb; 167:e230360.[3] T. Wakai, A. Mehregan, R. A. Fissore. Ca2+ Signaling and Homeostasis in Mammalian Oocytes and Eggs. Cold Spring Harb Perspect Biol. 2019 Dec; 11:a035162.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
