Advancing male age differentially alters levels and localization patterns of PLCzeta in sperm and testes from different mouse strains

Abstract

Sperm-specific phospholipase C zeta (PLCζ) initiates intracellular calcium (Ca2+) transients which drive a series of concurrent events collectively termed oocyte activation. Numerous investigations have linked abrogation and absence/reduction of PLCζ with forms of male infertility in humans where oocyte activation fails. However, very few studies have examined potential relationships between PLCζ and advancing male age, both of which are increasingly considered to be major effectors of male fertility. Initial efforts in humans may be hindered by inherent PLCζ variability within the human population, alongside a lack of sufficient controllable repeats. Herein, utilizing immunoblotting, immunofluorescence, and quantitative reverse transcription PCR (qRT-PCR) we examined for the first time PLCζ protein levels and localization patterns in sperm, and PLCζ mRNA levels within testes, from mice at 8 weeks, 12 weeks, 24 weeks, and 36 weeks of age, from two separate strains of mice, C57BL/6 (B6; inbred) and CD1 (outbred). Collectively, advancing male age generally diminished levels and variability of PLCζ protein and mRNA in sperm and testes, respectively, when both strains were examined. Furthermore, advancing male age altered the predominant pattern of PLCζ localization in mouse sperm, with younger mice exhibiting predominantly post-acrosomal, and older mice exhibiting both post-acrosomal and acrosomal populations of PLCζ. However, the specific pattern of such decline in levels of protein and mRNA was strain-specific. Collectively, our results demonstrate a negative relationship between advancing male age and PLCζ levels and localization patterns, indicating that aging male mice from different strains may serve as useful models to investigate PLCζ in cases of male infertility and subfertility in humans.

The mammalian sperm factor phospholipase C zeta is critical for early embryo division and pregnancy in humans and mice

“…Immunoblotting indicated a single band for PLCζ at 74 kDa in sperm from WT mice, while a single band was also observed in sperm from heterozygous of Plcζ mutant mouse sperm, but at a diminished intensity. Immunofluorescent analysis indicated the previously reported ( Kashir et al , 2021 ) fluorescence patterns in WT sperm, while sperm from Plcζ mutant mice exhibited a significantly diminished and dispersed pattern at the acrosomal region of the sperm head. Breeding experiments indicated a significantly reduced litter size of mutant Plcζ male mice compared to WT mice, while IVF-generated embryos using sperm from mutant Plcζ mice exhibited high rates of polyspermy, and resulted in significantly reduced numbers of these embryos reaching developmental milestones.…”

“…In line with such research, we have also shown recently that higher levels and specific localization patterns of PLCζ in human sperm are correlated with optimal ranges of sperm fertility parameters, as cases with higher proportions of successful fertilization exhibited such high levels or more physiological patterns of PLCζ ( Kashir et al , 2020 , 2023 ). Furthermore, we also demonstrated that such PLCζ parameters exhibited a negative relationship with advancing male age in mice, as the predominant pattern of PLCζ localization is altered and levels of PLCζ are generally diminished with advancing age (albeit in a strain-specific manner) ( Kashir et al , 2021 ). Collectively, such results suggest that PLCζ potentially represents a diagnostic measure not just of OAD, but also of a more general male population seeking fertility treatment.…”

SPERM FACTORS AND EGG ACTIVATION: Phospholipase C zeta (PLCZ1) and the clinical diagnosis of oocyte activation deficiency

“…However, the findings of such studies are very limited and further investigations are required before drawing firm conclusions ( Taylor et al 2010 , Park et al 2015 , Tavalaee et al 2017 ). Other studies have reported that the localization and total levels of PLCZ1 are not correlated with male age in humans although a recent study has reported otherwise in several mouse strains ( Yeste et al 2016 b , Kashir et al 2021 ). As discussed previously, it is clear that PLCZ1 is an important physiological factor that triggers oocyte activation.…”

The Therapeutic and Diagnostic Potential of Phospholipase C Zeta, Oocyte Activation, and Calcium in Treating Human Infertility

“…Sperm from oligoasthenoteratozoospermic, teratozoospermic, and asthenoteratozoospermic patients have been found to have lower levels of PLCζ [ 56 ]. Furthermore, sperm from globozoospermic patients usually exhibit a low rate of success in OA [ 104 ], either due to a lack of PLCζ, or if present at reduced amounts, they exhibit an abnormal localisation pattern [ 56 , 61 , 105 , 106 ].…”