Phospholipase C Zeta in Human Spermatozoa

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1		Alessandra Parrella	--	3525	2024-03-09 13:53:57	\|
2	Reference format revised.	Lindsay Dong	+ 3 word(s)	3528	2024-03-11 02:47:03	\|

This entry is adapted from the peer-reviewed paper 10.3390/ijms25021344

During fertilization, the fusion of the spermatozoa with the oocytes causes the release of calcium from the oocyte endoplasmatic reticulum. This, in turn, triggers a series of calcium ion (Ca²⁺) oscillations, a process known as oocyte activation. The sperm-specific factor responsible for oocyte activation is phospholipase C zeta (PLCζ). Men undergoing intracytoplasmic sperm injection (ICSI) with their spermatozoa lacking PLCζ are incapable of generating Ca²⁺ oscillation, leading to fertilization failure. The immunofluorescence assay is the most used technique to assess the expression and localization of PLCζ and to diagnose patients with reduced/absent ability to activate the oocytes.

PLCζ PLCZ1 human human spermatozoa human oocytes human infertility human oocyte activation

1. Introduction

Nowadays, infertility affects 8–12% of couples worldwide ^[1]. Of these, 20–30% can be attributed to the male, 20–30% to the female, 25–40% are related to both partners while 10–20% remain unexplained ^[2]. With the advancement of Assisted Reproductive Technologies (ART), such as ICSI and IVF, many couples with infertility issues can achieve pregnancy. ICSI is the most widely used procedure and represents a gold-standard technique to treat male infertility, particularly in cases with oligo-, astheno-, and teratozoospermia or with these combined factors. Additionally, ICSI can address issues related to the zona layer of the oocytes, such as zona pellucida and oolemma/ooplasmic abnormalities, or in other cases, such as elective oocyte cryopreservation, can address low oocyte maturity and recurrent polyspermy ^[3]. As a result, this technique now constitutes about two thirds of treatments while the IVF technique is used by only one third. Although IVF is an effective technique for many couples with infertility problems, its use is declining due to the high rate of total fertilization failure (TFF), which represents 5–10% of cases. This event, where oocytes fail to fertilize, remaining in metaphase II, occurs in 2–4% of cases when the ICSI technique is carried out and the main cause is largely believed to be oocyte activation deficiency (OAD) ^[1]^[4]. The key factor responsible for triggering oocyte activation is a sperm-specific factor, called phospholipase C zeta (PLCζ), which is localized in the perinuclear theca and promotes a series of Ca²⁺ oscillations in the oolemma that are crucial for the resumption of oocyte meiosis, pronuclear formation, and early embryo development ^[5]. The first study to prove the principal role of PLCζ in the oocyte activation events was conducted by Yoon et al. who demonstrated that spermatozoa with PLCζ deficiency in men undergoing the ICSI procedure were incapable of initiating the intracellular Ca²⁺ oscillation in the oocytes, leading to fertilization failure (FF) ^[6]. Since then, several studies have provided evidence of a strong association between impaired PLCζ functional ability and male infertility ^[7]. For instance, a study utilizing quantitative immunofluorescence analysis on a single sperm cell showed that the expression of PLCζ is diminished in infertile men when compared to fertile controls ^[8]. Furthermore, the same research team found a significant correlation between the total level and localization pattern of spermatozoa exhibiting PLCζ and the fertilization rate in men undergoing ICSI cycles ^[9]. The first genetic connection between OAD and PLCζ was documented by Heytens et al. with the discovery of a substitution mutation in an infertile male. This mutation was found at position 398 of the PLCζ open reading frame (ORF) in the Y domain, resulting in a histidine to proline substitution (PLCζ ^H398P) and leading to a disruption of the local protein fold ^[10]. Kashir et al. identified a secondary mutation within the X domain, involving the substitution of histidine with leucine at position 233 of the open reading frame (ORF). This mutation, designated as PLCζ ^H233L, is similarly accountable for the disruption of the local protein fold ^[11]. Recently, two more novel PLCζ mutations have been identified. One mutation, p.I489F, was identified in two infertile brothers and is located in the C2 domain. The other, p.S500L, a missense mutation, was observed in patients experiencing fertilization failure ^[12]. The current assessment to evaluate the expression of PLCζ is a quantitative immunofluorescence analysis. Kashir and colleagues have shown that there is a considerable variation in the total level of PLCζ not only between infertile and fertile men but also within different ejaculates of the same individual ^[8]. More tests have been developed to analyze the sperm activation potential in patients experiencing fertilization failure including heterologous and homologous assays such as the mouse oocyte activation test (MOAT) and the human oocytes Ca²⁺ analysis (H-OCA), respectively. These tests clarify the gamete responsible for OAD allowing a more specific and targeted treatment to achieve a higher fertilization rate in subsequent cycles, reducing the need for prolonged and potentially less successful interventions. Indeed, if the cause of fertilization failure is oocyte-related, a modified superovulation protocol should be applied. However, in the case of sperm-related OAD, the most suitable treatment option appears to be assisted oocyte activation (AOA) ^[3]. The commonly employed method for AOA involves the use of chemical activating agents such as ionomycin and calcimycin, which are able to trigger the meiotic resumption by raising intracellular Ca²⁺ levels. It has been shown by a variety of studies that, in couples with a history of <30% fertilization rate, the adoption of AOA in the subsequent ICSI cycle improves fertilization and pregnancy rates ^[3]^[13]^[14].

2. The Analysis of PLCζ

2.1. The PLCζ Mechanism of Action

PLCζ is a cytosolic sperm protein of 70 kDa with the unique ability to promote oocyte activation. It is composed of X and Y catalytic domains between which lies the XY-linker domain, four tandem EF-hand regions, and a C2 domain. The X and Y catalytic domains support the hydrolysis of phosphatidylinositol 4,5-biphosphate (PIP₂) while the high Ca²⁺ sensitivity is mostly due to the Ca²⁺ binding motifs EF-hands, which enable PLCζ to be active at basal Ca²⁺ levels in the egg cytoplasm. The XY-linker has a net positive electrostatic charge and therefore is involved in the interaction of PLCζ with the negatively charged substrate, PIP₂-containing membranes. Lastly, the C2 C-terminal domain of PLCζ is crucial for the function of PLCζ involved in targeting proteins to cell membranes. It has been shown that although the deletion of the C2 domain results in a partial loss of enzymatic activity, it has no impact on the enzyme’s Ca²⁺ sensitivity in vitro ^[15]. The mechanism of PLCZ signaling begins with the PLCζ diffusion in the ooplasm after the fusion of spermatozoa within the oocytes. Here, PLCζ binds intracellular vesicles containing PIP₂ and its hydrolysis results in the generation of inositol-1,4,5-triphosphate (InsP₃) and diacylglycerol (DAG). InsP₃ interacts with its receptor on the surface of the endoplasmic reticulum and promotes the release of calcium ^[7]. Infertile patients with PLCζ deficiency in their sperm are very likely to encounter IVF/ICSI failure. The quantitative analysis of PLCζ through immunofluorescence is a diagnostic marker to predict sperm oocyte activation capability. However, its clinical use is not always easy to apply because the inadequate specificity between the polyclonal nature of the antibodies and the masked PLCζ antigen causes poor PLCζ visualization and variability in the results. Various laboratories have tried to develop new protocols based on pepdite-blocking experiments ^[1].

2.2. PLCζ Level, Expression, and Localization in Relation to Semen Analysis

The characteristic localizations of PLCζ are in the equatorial, acrosome, post-acrosomal, and tail region of the spermatozoa, or a combination of these locations. However, the equatorial region is the most dominant localization ^[1]. A study has demonstrated that in men with normal semen parameters and a history of poor fertilization, the levels of PLCζ were low. The localization pattern was variable among these men, showing a low-intensity PLCζ distribution in the equatorial region, discontinuous patches along the equatorial band, and reactivity on the base of the head ^[16]. Similar results were found in a case report study in normozoospermic men with a history of complete fertilization failure. The PLCζ levels were decreased in the patient compared to the control, displaying an alteration of the localization of PLCζ. Indeed, the patient showed a significantly low percentage of spermatozoa with a localization pattern in the acrosome and the equatorial region but a significantly higher percentage in the midpiece. Also in the reacted acrosome, a significantly higher percentage was reported in the midpiece ^[17]. The same research group compared donors to normozoospermic patients and concluded that there was not a significant difference in the distribution patterns of PLCζ. A significant difference was seen when comparing donors to non-normozoospermic men, with a higher PLCζ acrosomal staining observed in donors. When concentration and motility were compared to PLCζ distribution patterns in donors and patients, no correlation was found between them ^[18].

2.3. The Level, Expression, and Localization of PLCζ and Fertilization Ability

Quantitative immunofluorescence analysis shows that total levels of PLCζ were significantly higher in fertile patients compared with infertile men diagnosed with recurrent ICSI failure. The localization pattern observed in each control and patient sample was significantly different. Spermatozoa from patients with OAD exhibited a punctate pattern of PLCζ localization, in contrast to the characteristic band observed in the equatorial, acrosome, and post-acrosome regions of spermatozoa in fertile men. Interestingly, total levels of PLCζ displayed a significant variance with the control showing levels like OAD patients ^[8]. Ferrer-Vaquer et al. also observed significant differences in PLCζ levels among control samples. Specifically, their analysis of PLCζ expression and localization in patients with low (<20%) or TFF revealed significantly lower levels of PLCζ compared to controls. It is worth noting that some control samples also exhibited levels similar to patients with FF, a trend that was similarly observed among the patients. They also examined sperm cells based on their acrosomal status, including intact acrosome, reacted acrosome, and unlabeled acrosome cells. In intact acrosome cells, PLCζ localization was primarily in the acrosomal region of most spermatozoa, with less extent in the equatorial or post-acrosomal regions. On the other hand, in cells with a reacted acrosome or with an unlabeled acrosome, PLCζ protein was displayed only in the post-acrosomal region ^[18]. The fertilization rates of ICSI and IVF procedures have been compared with PLCζ level and localization. Although no significant difference in PLCζ level and localization was observed in men undergoing IVF cycles compared to control, patients undergoing an ICSI procedure showed a significantly lower total level of PLCζ, with a lower percentage of total spermatozoa exhibiting PLCζ in the post-acrosomal and equatorial region and in the acrosomal + post-acrosomal + equatorial regions ^[9].

2.4. PLCZ1 Mutation Identified in Infertile Males

The human PLCZ1 gene is composed of 15 exons and it is situated on chromosome 12. In 2009, Heytens et al. first identified a genetic connection between OAD and PLCζ, pinpointing a substitution mutation in normozoospermic men ^[10]. The mutation, a histidine to proline substitution, arises in a Y domain of the active side of PLCζ at position 398 of the open reading frame (PLCζ ^H398P), resulting in the abolishment of the hydrolytic activity of PLCZ1 protein and the inability to generate Ca²⁺ oscillation in the ooplasm ^[1]. Subsequently, in the same patient, Kashir et al. identified a second mutation in the X domain resulting in a histidine to leucine substitution at position 233 of the open reading frame (PLCζ ^H233L), involved in the disruption of the local interaction within protein folding. This mutation does not eliminate PLCζ’s ability to generate Ca²⁺ oscillation, but its function is compromised. The author revealed that both mutations were heterozygous, with PLCζ ^H398P being inherited from the patient’s father and PLCζ ^H233L being inherited from the mother. This discovery was significant as it demonstrated for the first time that PLCZ1 can be inherited maternally, and that this could result in a loss of sperm function in the son and subsequently in infertility ^[11]. To verify the bilateral inheritance of the two mutations, the author analyzed the distribution of PLCζ ^H398P and PLCζ ^H233L as well as the localization patterns of fluorescent mutant PLCζ isoforms in human embryonic kidney cells (HEK293T) obtained from an infertile man with known PLCζ ^H398P and PLCζ ^H233L mutations. These mutations, located on different chromosomes, exhibit independent inheritance and are never present at the same time. Consequently, spermatozoa can only carry either the PLCζ ^H398P mutation or the PLCζ ^H233L mutation, but not both mutations simultaneously ^[19].

2.5. PLCζ in Globozoospermic Patients

Globozoospermia is a rare male infertility disorder in which spermatozoa have round heads, abnormal or absent acrosomes, and are often defective in two genes, DPY19L2 and SPATA16 ^[2]. In patients with globozoospermia, fertilization failure is caused by the absence or deficit of sperm oocyte activation factors such as PLCζ. Indeed, studies have shown that these individuals have a lower PLCζ mRNA, and protein level compared to fertile men ^[20]^[21]. This finding was also confirmed in a study of 32 globozoospermic men with DPY19L2 deletion, in which both the RNA and protein levels of sperm PLCζ were significantly lower than those in fertile men ^[22]. There are two subtypes of globozoospermia, the complete form (CG) and the partial form (PG), which are distinguished by the percentage of spermatozoa with round-headedness and acrosomal abnormalities. The complete form is the most severe and is characterized by 100% of spermatozoa having a round head and acrosomal hypoplasia; the partial form has a variable proportion of spermatozoa exhibiting round-headedness and acrosomal mark dysmorphism.

3. Phospholipase C Zeta in Human Spermatozoa

PLCζ is a soluble cytosolic sperm factor able to induce oocyte activation via the release of intracellular calcium ions from the endoplasmatic reticulum stores. A wide range of research has been conducted to investigate the role and function of PLCζ in male infertility, considered a causative factor in cases of fertilization failure. Researchers have investigated the PLCζ expression and localization in men with normal and abnormal semen parameters highlighting a strong correlation between sperm concentration, motility, and morphology with the percentage of spermatozoa positive for PLCζ. Notably, the latter plays a significant role in predicting PLCζ expression ^[23]^[24]^[25]. In contrast to these findings, Azad et al. reported no significant differences between polymorphic teratozoospermic patients and the control group in terms of the percentages of sperm that express PLCζ and their localization patterns. However, they did observe a significantly lower level of PLCζ expression among men with polymorphic teratozoospermia ^[26]. The same author shows a similar trend in oligoasthenoteratozoospermic men who displayed a significantly reduced percentage of sperm expressing PLCζ compared to normozoospermic men ^[23]. Another study performed by Ferrer-Vaquer et al. found no correlation between sperm characteristics and PLCζ expression in patient and donor samples, demonstrating that PLCζ expression might be independent from motility and concentration ^[18]. Rahimizadeh et al. recently investigated the possible connections between PLCζ levels and male age, sperm characteristics, DNA integrity, and cellular maturity in spermatozoa from men with either asthenoteratozoospermia or unexplained infertility. Although fertile men showed significantly higher levels of PLCζ than infertile or subfertile men and a correlation between PLCζ and the ability to bind hyaluronic acid was observed, no other associations could be identified ^[5]. Similar results were seen in another study where PLCζ immunoreactivity was not associated with the donor’s age, sperm concentration, motility, and DNA fragmentation. However, they found an inverse relationship with oxidative status ^[27]. On the contrary, Tavalaee et al. observed a significant negative correlation between DNA fragmentation and PLCζ expression, indicating that artificial oocyte activation may be required in males with high levels of DNA fragmentation ^[24]. The pattern of PLCζ localization in spermatozoa is a crucial consideration, as it has implications for both sperm health and the likelihood of reproductive success. A study has revealed that a dispersed PLCζ pattern is linked to lower sperm viability, while the presence of acrosomal + equatorial PLCζ is strongly associated with healthier sperm and successful fertilization. Variations in PLCζ patterns are correlated with declining sperm health, potentially contributing to male subfertility and the effects of advancing male age. Furthermore, in cases of successful fertilization, significantly larger amounts of PLCζ and higher ratios of equatorial and acrosome patterns were observed ^[25]. These results are consistent with previous studies reporting similar localization patterns ^[8]^[28]. A study on oligoasthenoteratozoospermic men showed significantly reduced proportions of the equatorial pattern and its combinations (equatorial + acrosome and equatorial + post-acrosomal) compared to the control group ^[23]. Interestingly, studies found that the total level, localization patterns and proportion of sperm exhibiting PLCζ are correlated with fertilization rates for the ICSI procedure ^[9]^[29]. Indeed, the mean percentage of PLCζ-positive sperm and the level of this protein were significantly decreased in FF patients compared to the control population ^[30]. A low expression of PLCζ has also been linked to both failed and low success in ICSI fertilization in normal-appearing sperm ^[16]^[17].Contrary to this, Aras-Tosun et al. demonstrated that the percentage and mean fluorescent intensity of the PLCζ protein do not exhibit a correlation with low fertilization and clinical pregnancy rates ^[31]. Nevertheless, there was significant variability in total PLCζ levels among individual controls. While patients with FF showed notably lower overall levels of PLCζ, the test results displayed variability, with certain controls exhibiting levels comparable to those found in FF samples ^[8]^[18]. This could be due to the lack of specific antibodies, making its implementation challenging. A novel methodology has been introduced to improve the visualization efficacy of PLCζ. This involves the utilization of an antigen unmasking/retrieval protocol, impacting both the relative fluorescence levels and the proportion of sperm exhibiting detectable PLCζ fluorescence ^[32]. On the other hand, Meng et al. demonstrated that their in-house method showed superior visualization and reliability to the unmasking/retrieval protocol ^[4]. Researchers have reported the inability of globozoospermic patients to evoke long-term Ca²⁺ oscillations because their sperm heads are rounded and devoid of the acrosome. These patients have an abnormal punctate pattern of PLCζ localization with some spermatozoa displaying an acrosomal bud. Moreover, the relative expression of PLCζ at RNA and protein levels is significantly lower compared to fertile men and therefore it is not surprising if these men are unable to fertilize naturally ^[20]^[21]^[22]. AOA is considered the best therapeutic option for these patients and for those suffering from OAD. Studies have shown that the addition of a calcium ionophore during the ICSI procedure can improve oocyte fertilization in men with a deficiency of PLCζ in their spermatozoa ^[33]^[34]^[35]. However, if the OAD is not derived from the male gamete, AOA may not always provide benefits. A recent study suggests that OAD can result not only from sperm-related factors but also from oocyte-related factors. Therefore, in cases where fertilization failure is attributed to oocytes, a modified superovulation technique should be considered ^[3]. Concurrently, in couples with sperm-related problems, various research laboratories are actively investigating new PLCζ-based techniques. Significant emphasis is being placed on sperm selection techniques to augment the percentage of spermatozoa exhibiting PLCζ. For instance, a study found that the use of density gradient centrifugation significantly decreases the proportion of sperm that did not express PLCζ removing the sperm with a low capability to induce oocyte activation ^[36]. Two sperm selection techniques have shown promise in increasing the expression levels of the PLCζ gene in sorted sperm. Microfluidic sperm selection effectively isolates sperm with elevated PLCZ1 expression levels. Meanwhile, the Zeta method enhances the intensity of PLCζ expression in selected spermatozoa, rather than increasing the number of spermatozoa showing PLCζ as seen with DGC. Therefore, it has been suggested that a good selection of spermatozoa with oocyte activation ability will be achieved with the combination of DCG and the Zeta method ^[37]. Studies have demonstrated that the MSOME technique can be beneficial in selecting spermatozoa that exhibit acrosome bud morphology and express PLCζ within their head regions. Specifically, up to 43% of spermatozoa with acrosome bud morphology have been found to express PLCζ ^[38]. Another approach is the microinjection of mRNA or recombinant protein in MII ^[7]. The injection of PLCZ1 RNA has been shown to induce the same pattern of Ca²⁺ oscillations as seen during fertilization, leading to the development of parthenogenetic blastocysts in humans ^[39]. However, it remains unclear whether the injection of hPLCZ1 RNA can cause any harm to human oocytes, despite its ability to produce optimal and physiological egg activation ^[13]. Nonetheless, the microinjection of pure hPLCZ could be a viable strategy for rescuing PLCζ and could be implemented immediately after an ICSI fertilization failure. This approach may be particularly beneficial for couples who have experienced repeated failures and have been advised to consider sperm donation ^[15]. Studies have found that genetic factors can contribute to PLCζ deficiency in male-related OAD. Many PLCZ1 gene mutations have been found in male individuals presenting low or total FF following the ICSI technique ^[11]^[12]^[19]^[40]^[41]^[42]^[43]^[44]^[45]^[46]^[47]^[48]. Most of these are homozygous PL CZ1 mutations, but it has been discovered that even if the heterozygous mutation is less common than homozygous mutations, they can decrease the overall quantity of functional protein in all sperm and be sufficient to result in FF following ICSI ^[12]. The impact of these mutations seems to depend on both their location within the PLCZ1 gene and their inheritance manner. It is probable that the deficiency of PLCZ1 is associated with unknown processes that are linked to gene expression or regulation ^[1]. Indeed, various processes can affect gene expression at the transcriptional and/or translational levels and identifying the specific process that impairs PLC function can be challenging. For instance, only a limited number of studies have examined the bidirectional promoter of PLCZ1, with Javadian-Elyaderani et al. being the only researchers to identify a variant in the CAPZA3 promoter located close to PLCZ1. However, the author hypothesized that this variant may not affect the transcription of PLCZ1 ^[41].

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Alessandra Parrella

Llanos Medrano

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María José Gómez-Torres

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Update Date: 11 Mar 2024

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