How do spermatozoa activation oocytes

The reason for this is the need for two constant and simultaneous active stimuli: cyclin B synthesis and cyclin-dependent kinase 1 CDK1. Swann et al. This factor remained unidentified for several years before indirect evidence in literature helped define it.

A timeline with the most important reports about the role of the sperm factor candidates in the oocyte activation mechanism. This mechanism should involve the increased production of IP 3 , regulated by the phosphoinositide-signaling pathway. The sperm head has a region called the perinuclear theca PT , a condensed cytosolic protein layer that surrounds the nucleus and can be divided into structural or functional zones [ 29 ].

The functional zones are divided into three further parts: a subacrosomal region, equatorial segment, and postacrosomal sheath-perinuclear theca PAS-PT. Experiments assessing the regions of the sperm capable of activating oocytes, involved an injection of the head or tail of a spermatozoon into mouse oocytes [ 30 ], indicating that only the sperm head can activate the oocyte. Moreover, when sperm heads were treated with substances that alter all membranes, such as proteases or detergents that denaturalize proteins, sperm failed to activate the oocytes.

Meanwhile, when treated with Triton X, a non-ionic surfactant that removes all membranes except the PT around the nucleus, the sperm retains the ability for activation [ 31 ]. Data show that SOAF could be a protein [ 28 , 32 ]; however, most of the proteins investigated did not match the physiological characteristics expected of an oocyte activator factor.

There are no scientific reports of its function in mammalian oocyte activation or in the fertilization process [ 36 ]. As the PLC family of proteins has a critical function in the oocyte activation cascade, they became a major focus of clinical research [ 14 , 40 ]. This family of PLCs currently has 13 isozymes [ 43 ]. The structure and functional domains of the SOAF may play an important role [ 57 ].

SOAFs protein scheme. However, this unknown SOAF in the human sperm has not yet been confirmed [ 61 , 62 ]. It is located in the perinuclear matrix of the sperm head Fig. In , Wu et al. Other independent groups have demonstrated the association between PAWP expression and the competence of human and bull sperm [ 65 , 66 ]. Mehlmann et al. However, this mechanism requires further research, Aarabi et al.

These epigenetic events affect early embryo development, and cause phenotypic changes in the offspring [ 70 ]. During fertilization, sperm DNA compaction and protamine replacement are the most important events to form the zygote nucleus, while ncRNAs are involved in embryonic development and transgenerational adaptation [ 69 ].

Sperm chromatin includes testicular histones, protamines, and free DNA, called nuclear matrix. The complex transition between the oocyte and the spermatozoon chromatin structure is poorly understood, but it is known that in the spermatozoon, the protamine incorporation during spermatogenesis, as well as its removal upon fecundation are, apparently, critical for paternal epigenetics profile and reprogramming.

In contrast to the passive demethylation that occurs in the oocyte DNA upon fertilization, the sperm chromatin undergoes throughout deprotamination followed by dramatic demethylation and active decondesation of the chromatin, creating an environment that facilitates a new methylation profile, thus new epigenetics marks, suggesting that determination of cellular fates for tissue specification is critical [ 73 , 74 , 75 , 76 ].

Although the presence of different types of ncRNAs in the sperm has been demonstrated, the function of these during fertilization is not clear [ 69 ]. After fusion of the sperm with the oocyte membrane, pronucleus formation occurs. Lack of pronuclei is a clear sign of failed fertilization during ICSI. However, a percentage of ICSI cycle failure has been reported [ 77 ], even with good ovarian response and semen quality.

As the spermatozoon is already within the oocyte, the fertilization failure could be associated with sperm factors or oocyte activation machinery. These findings strongly suggest that this mutation can cause male infertility in humans. Another genetic pathology known as globozoospermia is associated with the capability of activating oocytes.

This autosomal-recessive pathology, which can be partial or total, is commonly caused by mutations in the DPY19L2 gene involved in developing the acrosome and elongation of the sperm head; therefore, the spermatozoon lacks the acrosome or shows an abnormality in other structures that provide its characteristic shape [ 8 , 50 ].

Less common causes of TFF are sperm head decondensation, premature sperm chromatin condensation, oocyte spindle defects, and sperm defects [ 86 ].

Ferrer-Buitrago et al. ARTs involve ovarian stimulation, gamete and embryo manipulation, and cryopreservation. At present, IVF and ICSI are reproductive technologies that are widely used to treat infertility related to reproductive endocrinology, genetic disorders, oocyte donation, and surrogacy.

The aim of ART is to attain a successful pregnancy, and during this process, most biological barriers are bypassed, especially when ICSI is applied, because a morphologically normal spermatozoon is directly injected into a mature oocyte [ 7 ]. Since the early s, when the first pregnancy using ICSI was reported [ 88 ], almost any type of spermatozoa were used to fertilize an oocyte. Today, the clinical situation has improved with research, allowing the investigation and determination of gene expression, proteins, and molecular pathways related to gamete formation and development, fertilization processes, and embryo development.

The lack of pronuclei formation after a conventional IVF procedure is a clear sign of fertilization failure. The reasons can be multiple, such as non-recognition between the spermatozoon and the oocyte or failure of acrosome reaction, among others. Applied, for example, when oocytes are cryopreserved or in severe male infertility cases. Although there is controversy, there are two main sources of potentially usable sperm, testicular or epididymal spermatozoa obtained through biopsies or ejaculated sperm.

While the protamine content in the ejaculated spermatozoa confers the ability to be reprogrammed after fertilization [ 89 ], thus the ejaculated sperm is thought to be more mature; it has been proposed that the extraction of testicular sperm may eliminate the exposure to the reactive oxygen species and could result in the access to high-quality spermatozoa [ 90 ].

However, most cases are successful, but there is a small percentage of cases in which conventional ICSI fails.

The mechanisms that are altered are unknown, and researchers are currently uncertain of solutions. In most cases, other complementary strategies, such as chemical adjuvants or alternative techniques, are applied to improve fertilization and achieve better embryo development. Localization patterns of proteins, receptors, DNA, and membrane integrity help elucidate the stages in oocyte activation and reasons for failures; they also help to develop diagnostic tests and therapeutic strategies to restore fertilization, either through pharmaceutical agents or reproductive technology or both.

Therefore, to activate oocytes, the most common protocol applied in humans includes the ready-to-use ionophore A, following ICSI [ 18 , 96 ]. AOAs have been used for over a decade in reproductive medicine.

Many reports have shown the beneficial effects of AOAs in reproductive medicine in couples with TFF [ 18 , 97 ], male patients with severe sperm alterations including globozoospermia [ 98 , 99 ], teratozoospermia [ 80 , ], cryptozoospermia, azoospermia [ ], and sperm stress conditions, such as cryopreservation protocols [ , ].

In , Murugesu et al. Reports using AOAs in an ICSI cycle are contradictory, so it is possible that only a subset of patients will benefit from them [ ].

Concern still exists regarding the potentially deleterious effects of these substances on embryogenesis [ ]. Vanden Meerschaut et al. This group reported no severe effects in the offspring. However, the high response rate and the robustness of the test used in this study are still considered preliminary because the sample size was small. Another important problem is the introduction of genetic material into the oocyte, which is forbidden for human medicine in most parts of the world.

Still, its application in IVF clinics is limited because of commercial availability. Authors have proposed that routine sperm preparation methods, including density gradient selection or swim-up selecting sperm by motility and morphology , be modified and include other selected molecular or cellular sperm characteristics. One example is selecting for surface markers using magnetically activated cell sorting MACS for the selection of apoptotic sperm, which express phosphatidylserine in their membrane [ ].

These modifications of preparation methods could improve sperm selection and improve fertilization treatments. Thus, Chan et al. In accordance, Khakpour et al. This lack of response was also observed in cows and pigs [ 61 , ]. However, Norozi-Hafshejani et al. These strategies enable solutions for infertility problems related to the spermatozoon, such as globozoospermia or recurrent ICSI cycle failure because of OAD.

However, when sperm preparation procedures target some characteristics of the spermatozoon, such as DNA fragmentation levels or acrosome and membrane integrity, modifications of the preparation method must be chosen with care [ ]. Since Jacques Loeb proposed that the spermatozoon plays a key role in fertilization, more than just providing genetic material [ 11 ], a new field in reproductive biology opened a series of investigations and debates on the role of the male gamete in early embryo development and its effects on the offspring.

This resulted in the knowledge of spermatozoon factors as fundamental to initiating oocyte activation, and the sperm epigenome important role for successful embryogenesis. Many factors and molecular pathways have been studied to determine pronuclear formation, for which the oocyte machinery modifies the sperm chromatin structure after fertilization.

Several mammals, as well as heterologous ICSI, have been used as model systems to study oocyte activation. Among these, similar observations between human and mouse oocyte fertilization and activation mechanism and embryo development processes allow us to compare and contrast the details [ 9 , 84 , 92 , , , , , ].

The development of ART, especially ICSI techniques that bypass any oocyte-sperm membrane-binding mechanism, concluded with a soluble SOAF that enters the oocyte upon fertilization, surviving to the acrosome reaction.

Since all IVF clinics do not have access to animals for assays, particularly mouse models to evaluate heterologous ICSI and the ability of the human sperm to activate mouse oocytes, a standardized protocol should be developed. This protocol must have all necessary biochemical characteristics, including analytical and biological limits, a scientific validation, reference values or range, normal variation, and a consensus of the professional community when alterations are observed [ ].

Thus, treatments are empirical since not all patients respond to AOA treatments and each IVF clinic has its own protocol for such cases. Alterations in gamete development or the fertilization process show that embryogenesis may fail or that multiple disorders could appear in offspring. In human reproduction, maternal, paternal, or idiopathic factors could cause this. Mammalian phospholipase Czeta induces oocyte activation from the sperm perinuclear matrix.

The pattern of localization of the putative oocyte activation factor, phospholipase C zeta, in uncapacitated, capacitated, and ionophore-treated human spermatozoa. Hum Reprod ; 23 : — Mol Hum Reprod ; 21 : — Biochem J ; : — Sperm-borne protein, PAWP, initiates zygotic development in Xenopus laevis by eliciting intracellular calcium release.

Mol Reprod Dev ; 77 : — Swann K , Lai FA. Cell Calcium ; 53 : 55 — Hum Reprod ; 26 : — J Clin Invest ; : — Reduced amounts and abnormal forms of phospholipase C zeta PLCzeta in spermatozoa from infertile men.

Hum Reprod ; 24 : — Hum Reprod ; 27 : — PLCz functional haplotypes modulating promoter transcriptional activity are associated with semen quality traits in Chinese Holstein bulls. PLoS One ; 8 : e Protein expression pattern of PAWP in bull spermatozoa is associated with sperm quality and fertility following artificial insemination.

Mol Reprod Dev ; 81 : — Biol Reprod ; 72 : — PLoS One ; 7 : e Fertil Steril in press. Published online ahead of print 12 June ; DOI Artificial oocyte activation intracytoplasmic sperm injection. Fertil Steril ; 94 : — Recombinant human phospholipase C zeta 1 induces intracellular calcium oscillations and oocyte activation in mouse and human oocytes.

Species-specific differences in the activity and nuclear localization of murine and bovine phospholipase C zeta 1. Biol Reprod ; 83 : 92 — Motile sperm organelle morphology evaluation-selected globozoospermic human sperm with an acrosomal bud exhibits novel patterns and higher levels of phospholipase C zeta. Fertil Steril ; 99 : — Zanetti N , Mayorga LS. Acrosomal swelling and membrane docking are required for hybrid vesicle formation during the human sperm acrosome reaction.

Biol Reprod ; 81 : — Mol Hum Reprod ; 20 : — Asian J Androl ; in press. Published online ahead of print 23 January ; DOI: Arrest of spermatogenesis at round spermatids in PLCz1 deficient mice. Universal Protein Resource [Internet].

Accessed 18 March Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide.

Sign In or Create an Account. Sign In. Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Calcium Signaling upon Fertilization. The SOAF. Phospholipase C Zeta. Oxford Academic. Marc Yeste. Celine Jones. Kevin Coward. Revision received:.

Select Format Select format. Permissions Icon Permissions. Abstract The beginning of embryogenesis is preceded by a sequence of events mediated by the release of intracellular calcium in the ooplasm, a multifaceted process known as oocyte activation. PAWP a. Study year [reference] b. Expression of PAWP is restricted to the elongating spermatids and thought to be secreted later by the epididymis.

Saunders et al. The 74 kDa active isoform was found only in the tail fraction arrow. Lanes were run on the same gel but were non-contiguous. C Human sperm extraction by NP The functional isoform arrow was detergent extractable as shown in NP40 lane. WS, Whole sperm; Sc. Sn, Sonication supernatant. However, the 74 kDa band and most likely its breakdown product, a 55 kDa protein band, were extracted by NP40, while the detergent insoluble 45 kDa band remained in the pellet Fig.

We performed immunoproxidase staining on mouse and human paraffin embedded testicular tissues. The immunoreactivity was concentrated in the acrosomic granule, located at the center of AV Fig. Surprisingly, by step 14 spermatids stages II and III of spermatogenesis , the intensity of immunostaining associated with elongated spermatid nuclei diminished substantially.

A Immunostaining originates at the beginning of acrosome formation. In stage III it appears confined to the acrosomic vesicle AV of step 3 spermatids as seen in detail in the inset; note that acrosomic granule is most immunoreactive. In stage VII immunostaining is intense within the acrosome, capping a portion of the nucleus arrowheads of the step 7 round spermatids as seen in more detail in the inset.

In stages XI and XII immunostaining resides in the head of elongating spermatids in steps 11 and 12, respectively arrows. However, by step 14 stages II and III the intensity of immunostaining in elongated spermatid heads has diminished significantly arrows.

Little immunoreactivity is found elsewhere in the epithelium and the elongated spermatids arrowheads appear unreactive. In step 4 spermatids stage IV, see C , the fully formed acrosome is intensely labeled arrows. In step 4 spermatids stage IV immunostaining was concentrated over the fully formed acrosome, covering the apical half of the spermatid nucleus. Importantly, little immunoreactivity was found elsewhere in the epithelium and elongated spermatids Fig. In both species, saturation of antibodies with oligopeptides used to raise the immune serum resulted in elimination of immunostaining over the round spermatids of seminiferous tubule sections in similar stages of development, confirming the specificity of reactions Fig.

Throughout the last steps of spermiogenesis there was a noticeable diminution in the intensity of acrosome labelling Fig. Similar results were obtained by anti-EF. B By step 2, several immunoreactive proacrosomic granules have fused with each other forming the acrosomic vesicle AV , which attaches to the spermatid nucleus N. C Section through a portion of step 3 spermatid showing the immunogold labeled AV docked onto the nucleus with the subacrosomal layer SL intervening.

Underlying the nuclear envelope in the region of the forming acrosome is the electron dense nuclear lamina outlined by asterisks. D As acrosome capping proceeds over the apical half of the nucleus in step 6 spermatids a high concentration of immunogold labeling is evident in the expanding acrosomal collar AC coincident with a loss of labelling in the acrosomic granule. E Step 16 spermatids, almost fully formed, show a diminution of labeling in the acrosome and no labeling is detected in the perinuclear theca.

As shown in Fig. Comparing permeabilized vs. B, C Mature spermatozoa were extracted from cauda epididymis and ejaculate in mouse and human, respectively. Furthermore, we performed in situ hybridization to localize the mRNA in the epididymal epithelium. Inset shows perinuclear localization of mRNA within the principal cell.

The interstitium within the inset is marked by asterisk. No signal is detected in the corresponding regions of control probe right panel. C Paraffin embedded mouse epididymal tissues immunoproxidase stained with anti-EF antibody. Right panel shows the result of pre-incubating the primary antibody with the oligopeptides used to raise the immune serum.

The principal cells within initial segment of the caput epididymis were found to be most immunoreactive. Within the epididymal lumen, immunostaining was most evident in sperm tail and cytoplasmic droplet not shown. Subsequently, during the zona-induced acrosome reaction, the immunoreactivity accompanied the acrosomic shroud and was lost from the sperm head. The immunoreactivity disappears along with acrosome shroud arrows when the spermatozoa undergo the acrosome reaction.

During fertilization, motile and fertilization competent spermatozoa pass through the female reproductive tract, interact with the zona pellucida and activate the metaphase II arrested oocytes after sperm-oolemma fusion [34].

The ability of spermatozoa to activate oocytes depends upon the normal expression of the sperm borne oocyte activating factor SOAF during spermiogenesis and its appropriate assembly as part of the PAS-PT [9] , [10]. Therefore, it is crucial to identify SOAF and its potential contribution to infertility, as well as to harness it for infertility treatments. Grasa et al. It was not retained in the sperm head as expected but rather in the sperm tail.

Young et al. However, they didn't further analyze whether the 74 kDa band belonged to the sperm head or tail. Similar to our findings in mouse, Bedford-Guaus et al. The sperm heads and tails were separated by sonication and centrifugation in this study, while no detergent extraction was performed. First, we injected either a single mouse spermatozoon or round spermatid into a mature mouse oocyte. As has been already reported [ 29 , 32 ], mouse spermatozoa successfully activated homologous oocytes, but round spermatids and primary spermatocytes did not Table 1.

As shown in Figure 1a , an oocyte that received a mouse round spermatid stayed at M II and induced premature condensation of the spermatid chromosomes.

Thus, we confirmed that our injection technique was reliable enough to be applied to the next series of experiments using monkey spermatozoa and spermatogenic cells.

No oocytes were activated by monkey lymphocytes Table 1. There was a marked tendency for oocytes activated by monkey spermatozoa or spermatogenic cells to form a single pronucleus as compared with those activated by mouse spermatozoa Table 1.

Activation and pronuclear formation in mouse oocytes after injection with mouse or monkey male germ cells. PN, Pronucleus; PB 2 , second polar body.

The oocyte remained arrested at M II and the spermatid chromosomes underwent premature condensation arrow. F, Female chromosomes. The oocyte was activated and formed a spermatid-derived male pronucleus M and a female pronucleus F. Cynomolgus monkey epididymal spermatozoa a and spermatogenic cells b.

Monkey spermatogenic cells can easily be identified by their morphology. Arrows and arrowheads in b indicate spermatocytes and round spermatids, respectively.

The zero time is the time of injection, and the recording was started about 8 min later. This pattern was reproduced in the present experiments Fig. Seventeen of the 25 oocytes were subjected to nucleus staining, and the nuclear stage could be determined in 14 of 17 oocytes. To examine SF in the oocytes that were activated by sperm or spermatogenic cells, we transferred their karyoplast, cytoplast, or second polar body into freshly recovered unfertilized oocytes by electrofusion.

The male and female pronuclei, but not the second polar body or the cytoplast, from oocytes activated by mouse spermatozoa induced activation in most recipient oocytes. Some of the cytoplasts at T II activated recipient oocytes, indicating that SF was distributed throughout the cytoplasm shortly after fertilization.

Here again, spermatocytes have less, if any, oocyte-activating capacity as compared with more mature sperm cells. In every experimental group, there was a tendency for the male pronuclei to show better oocyte-activating capacity than the female pronuclei or cytoplast.

Oocytes that proceeded to T II or further at 2 h after transfer were considered to be activated. This study supports the notion that mouse oocytes are a useful model for assessing SF in male gametes. Using this experimental system, we demonstrated that monkey spermatozoa readily activated mouse oocytes and formed a well-developed male pronucleus at a rate similar to homologous fertilization.

This study also revealed that monkey round spermatids that have not commenced spermiogenesis have full oocyte-activating capacity, like mature spermatozoa. This contrasts with mouse round spermatids that completely lack this capacity [ 12 , 29 , 32 ]. Thus, the stage at which SF first appears or becomes active depends on the species, although the SF activity is not strictly species specific, as has been demonstrated by previous heterologous experiments [ 4 — 10 , 12 ].

The presence of active SF at the round spermatid stage has been reported only in humans [ 11 ]. This indicates that human and monkey round spermatids have already matured as spermatozoa in the sense of oocyte-activating capacity. In fact, normal babies have been born in human clinics following intracytoplasmic injection of round spermatids without the aid of artificial stimuli to activate oocytes [ 35 ].

The possibility that oocytes might have been stimulated by the medium coinjected with monkey spermatocytes can be excluded because injection of primary spermatocytes from mice did not eventually activate oocytes see Table 1.

Very recently, Yazawa et al. Whether the monkey SF is really expressed in spermatocytes will be determined more precisely when the SF molecule is identified. In this study, a considerable number of mouse oocytes injected with monkey spermatozoa or spermatogenic cells formed a single pronucleus.

According to Rybouchkin et al. Therefore, it is most probable that the chromosomes from monkey spermatozoa or spermatogenic cells intermingled with mouse chromosomes to form a single chromosome mass, rather than failing to be decondensed. Our nuclear transfer experiments using mouse oocytes activated by mouse spermatozoa confirmed the previous study by Kono et al.

We further showed that the oocyte-activating activity exists in the ooplasm shortly after fertilization T II but is absent in the second polar body. These findings suggest that SF diffuses over the ooplasm from the incorporated or injected spermatozoon and then is concentrated to the pronuclei after the second meiotic division. It will be interesting to examine whether those dynamics of SF are common in mammalian species. The present study showed that the SF liberated from the injected monkey spermatozoa or spermatids is capable of directing to the pronuclei in mouse oocytes, as the mouse SF.

The oocyte-activating activity remained in the ooplasm in some cases, suggesting that some species specificity may exist in the process of concentration of SF into the pronuclei see Table 2.

Thus, it is probable that each component the pronuclei, cytoplast, and second polar body contained only a part of the total monkey SF introduced into oocytes, leading to lower activation rates after nuclear transfer than the pronuclei of oocytes injected with mouse spermatozoa. Some pronuclei and cytoplasts from oocytes injected with monkey primary spermatocytes activated recipient oocytes, but the rate was not significantly different from that of the control nuclear transfer using strontium-treated oocytes.

Although the biological and biochemical properties of mammalian SF have been studied extensively, the SF molecule is not identified yet. When the SF protein is identified, its expression during spermatogenesis, its species-specificity in the oocyte-activating capacity, and its localization in activated oocytes will be determined in a more direct, precise manner.

Swann K. Rev Reprod 1 : 33 — Google Scholar. Dev Biol : 62 — Tesarik J , Sousa M. Fertil Steril 62 : —