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Disruption of gamete fusion alters the sperm-egg ratio at gamete interaction

Journal of Translational Medicine volume 22, Article number: 1134 (2024) Cite this article

Abstract

Background

The mechanisms enabling sperm to locate unfertilized eggs within the fallopian tubes remain a subject of debate in reproductive biology. Previous studies using polytocous mammals observed a 1:1 sperm-egg ratio within the ampulla at the time of fertilization. From these observations, it is hypothesized that this mechanism could be linked to sperm-egg fusion, such that unfertilized eggs may attract sperm until fusion occurs, whereupon the attraction ceases.

Methods

To test this, fertile male mice were mated with infertile homozygous Cd9Null females, whose eggs cannot fuse with sperm, leading to the accumulation of supernumerary sperm in the perivitelline space. Fertile heterozygous Cd9Het females, were used as controls.

Results

The results revealed that both Cd9Null and Cd9Het females ovulated similar numbers of eggs (6.53 ± 0.61 vs. 5.50 ± 0.53 eggs/ampulla). The majority of eggs produced by Cd9Het females were fertilized by one single sperm, without any additional sperm found bound to the zona or within the perivitelline space. In contrast, most of the eggs ovulated by Cd9Null females either showed an accumulation of supernumerary sperm within in the perivitelline space or showed no sperm bound to the zona nor present within the perivitelline space.

Conclusions

These findings indicate that genetic ablation of Cd9 leads to an imbalance in the 1:1 sperm-egg ratio observed within the ampulla. This information may set the foundation for future studies with the aim to identify the specific mechanisms that sperm use to locate unfertilized eggs and whether they become ineffective when gamete fusion is prevented.

Background

In mammalian reproduction, a large quantity of sperm enter the female reproductive tract, yet few manage to reach the oviduct and find an oocyte. Sperm in the oviduct bind to the oviductal epithelium of the isthmus, forming a sperm storage reservoir [1,2,3]. Within this reservoir, sperm undergo capacitation—a process that enables sperm to fertilize the oocyte [4, 5]. Capacitation occurs asynchronously, and only up to 10% of spermatozoa are capacitated, reducing the number of capacitated sperm in the ampulla to approximately 10–20 cells [6,7,8]. Given the minute size of the gametes (~ 120 μm) relative to the oviductal dimensions, the likelihood of incidental arrival of sperm at the oocyte is minimal. This leads to the hypothesis of the presence of guidance mechanisms that direct the sperm from the isthmus through the ampulla and the cumulus oophorus, before sperm bind the extracellular zona pellucida surrounding the egg [9, 10].

Sperm of non-mammalian species such as echinoderms [11], insects, and fish [12, 13], use specific signaling mechanisms to locate unfertilized eggs. Although it remains uncertain whether mammalian sperm employ similar signaling mechanisms for egg localization, investigations in mice, rats, and pigs demonstrate that individual spermatozoa can successfully localize and fertilize each egg within the ovarian ampullae [14, 15]. During normal mating, most eggs within the cumulus oophorus of polytocous mammals are fertilized within a relatively brief period of time [16]. Indeed, effective localization of unfertilized eggs is important for successful fertilization, as delayed fertilization can negatively impact preimplantation embryonic development [17, 18].

Sperm infrequently reverse their direction of motility once interacting with the zona pellucida, therefore, it is suspected that molecular mechanisms that have yet-to-be-identified facilitate the guidance of sperm towards unfertilized oocytes, to ensure that each egg interacts with at least one sperm. Also, numerous pioneer studies in polytocous mammalian species (e.g., mice, rats, rabbits) report a 1:1 sperm-egg ratio within the ampulla at the time of fertilization [7, 8, 19,20,21,22,23]. These observations may be explained by random effect — due to tens of sperm being present within the ampulla [24], one single sperm may find one unfertilized egg by random chance. Alternatively, a signaling mechanism may mediate the attraction of one sperm to one unfertilized egg; such a mechanism may cease once the egg has fused with the sperm. At present, it remains uncertain whether the ‘1:1’ sperm-to-egg ratio is influenced by gamete fusion or by random effect. If unfertilized eggs possess a mechanism to attract sperm that ceases upon gamete fusion, then the remaining sperm would redirect to locate unfertilized eggs that are still capable of sperm attraction. This model predicts that preventing gamete fusion may lead to an accumulation of an excess number of sperm gathering around a small number of eggs, resulting in the majority of eggs not interacting with any sperm. After binding and crossing the zona pellucida, sperm adhere to the egg plasma membrane, also known as the oolemma. After successful adhesion, sperm fuse with the oolemma. CD9, is a tetraspanin protein expressed in the oolemma and was the first one identified as necessary for gamete adhesion prior to fusion [25]. Females lacking CD9 exhibit significantly reduced fertility or infertility due to impaired sperm adhesion to the oolemma, resulting in supernumerary sperm accumulating within the egg’s perivitelline space [26]. In this study, we use Cd9Null mice to investigate sperm-egg ratios following spontaneous mating in mice and we explore the role of gamete fusion in monospermic interactions with ovulated oocytes.

Methods

Genotyping

Transgenic Cd9Null mice were genotyped using PCR with the following cycle conditions: [95 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min] × 30 cycles, followed by 72 °C for 7 min, then storage at 4 °C for > 30 min. This was performed using primers from previous studies [26].

Light microscopy

Samples were mounted in PBS, and images of oocytes and embryos were acquired using a confocal microscope LSM 800 (Zeiss) with 10X or 20X objectives, at room temperature. LSM 800 images were processed with ZEN 3.2 software (Zeiss), exported as 300 dpi resolution TIFF files and compiled using Adobe Illustrator 25.0. Maximum intensity projections of confocal optical sections were generated and combined with DIC images of oocytes, embryos .

Mating and gamete collection

Female mice used in the experiment were allowed to undergo their natural ovulation cycle without artificial stimulation. Homozygous and heterozygous Cd9 KO female mice (with an outbred genetic background) were co-mated overnight with sexually mature ICR-CD1 males, aged 12–14 weeks, confirmed to be fertile based on their ability to sire offspring. Eight independent mating experiments were performed. After mating, females that displayed a copulatory plug were euthanized and oocytes were collected from the ampullae. After images were acquired, the oocytes of the homozygous Cd9 KO (Cd9Null) females were evaluated for the presence of sperm within the perivitelline space and the oocytes collected from the of heterozygous Cd9 KO (Cd9Het) females were evaluated to count the number of one- and two-cell embryos.

Gamete or embryo quantifications and statistics

Sperm bound to oocytes or located within the perivitelline space, as well as the number of fertilized eggs and one- and two-cell stage embryos, were quantified using maximum intensity projections acquired through confocal microscopy (LSM800, Zeiss). Presence of sperm within the perivitelline space or the formation of embryos from gamete fusion were detected and analyzed using the ZEN 3.2 software (Blue Edition, Zeiss), and descriptive statistics were used to analyze number of sperm/oocytes or sperm/embryo using One-Way ANOVA, followed by the Tukey’s Honestly Significant Difference (HSD) post-hoc tests. Sperm counts (n = 5 experiments) were shown as boxplots with error bars indicating data points within the 10th and 90th percentiles and a horizontal line representing the median. The middle two quartiles were shown as boxplots, and bold points were used to mark outliers. All statistical analyses were conducted using R Studio (v. 3.6.3).

Results

Studies in mice, rats, and rabbits report a 1:1 sperm-egg ratio within the ampulla at the time of fertilization [7, 8, 19,20,21,22,23]. These observations may be explained as a random effect — such that, due to the presence of hundreds of sperm within the ampulla, a single sperm may find one unfertilized egg by random chance. Alternatively, a yet-to be identified signaling mechanism may mediate the attraction of one sperm to one unfertilized egg, which would, in turn, facilitate in the redirection of sperm away from the fertilized eggs and encourage sperm interaction with the remaining unfertilized eggs. One could argue that this putative mechanism may be dependent on sperm-egg fusion, such that unfertilized eggs attract sperm until gamete fusion, whereupon this mechanism ceases in function. Therefore, the remaining sperm would find other unfertilized eggs. This model raises the prediction that preventing gamete fusion may result in the gathering of supernumerary sperm to a small number of unfertilized eggs, resulting in the majority of unfertilized eggs without sperm.

To test this hypothesis, fertile male mice were mated with Cd9Null females, whose eggs cannot fuse with sperm, accumulating supernumerary sperm within the perivitelline space [26]. Fertile Cd9Het females were used as the controls. Males mated normally with naturally ovulated female mice; 16 h post mating, we collected ampullae from female mice showing a clear copulatory plug and uterine horns filled with sperm — which are clear indicators of successful copulation. Unfertilized and fertilized eggs were collected and stained with WGA 633 and Hoechst to detect zona pellucida and sperm, respectively.

Under natural ovulation, Cd9Null and Cd9Het females ovulated a comparable number of MII eggs (6.53 ± 0.61 vs. 5.50 ± 0.53 eggs/ampulla)(Fig. 1A). To quantify the eggs presenting a ‘one-egg to one-sperm’ ratio, for the Cd9Null, we counted the number of eggs presenting one single sperm in the perivitelline space; and for the Cd9Het, we counted the number of fertilized eggs (2-cell embryos) presenting no sperm in the perivitelline space (Fig. 1A-C). The majority of Cd9Het eggs (78.54 ± 2.98%; s.e.m) were shown to be crossed by one single sperm, which is consistent with previous studies [7, 8, 19,20,21,22,23, 27], while the remaining eggs were either shown to have not been crossed by sperm (10.15 ± 5.13%; s.e.m) or to have been crossed by multiple sperm at 2–3 sperm/egg (11.31 ± 2.89%; s.e.m). By contrast, the majority of Cd9Null eggs (54.42 ± 7.81%; s.e.m) were shown to have been crossed by no sperm, while the remaining eggs were either shown to have one single sperm within the perivitelline space (10.15 ± 5.11%; s.e.m) or to have accumulated a supernumerary quantity of sperm within the perivitelline space at 2–14 sperm/egg (34.79 ± 7.05%; s.e.m) (Fig. 1C and D).

Fig. 1
figure 1

Sperm-egg interactions inCd9NullandCd9Hetfemales. (A) Quantification of naturally ovulated MII oocyte from Cd9Null and Cd9Het females (n = 16). Boxplots represent the median (vertical line) number of ovulated oocytes, with data points within the 10th and 90th percentiles shown as error bars. Boxes encompass the middle two quartiles, and dots indicate outliers. Superscript letters denote statistical significance (P < 0.05) as determined by One-way ANOVA followed by Tukey’s HSD (honestly significant difference) post-hoc test. (B) Schematics showing different possible scenarios for sperm-egg ratio in the presence or absence of gamete fusion. (C) Maximum intensity projections of confocal optical sections to a single plane combined with DIC images of MII oocytes, fertilized eggs or embryos collected after in vivo mating, presenting no sperm, 1 sperm, or more than one sperm per oocyte. Yellow, WGA-633; cyan, sperm nuclei stained with Hoechst. (D) Same as in A, for observations shown in (C)

Full size image

Therefore, it is reasonable to conclude that the 1:1 sperm/egg ratio observed in the ampullae of fertile females is dependent on gamete fusion. This evidence suggests the existence of mechanisms for sperm to locate unfertilized eggs within the ampulla.

Discussion

This study shows that Cd9Null oocytes collected from naturally ovulated females mated with fertile males exhibit either an absence of sperm or an excessive accumulation of sperm in the perivitelline space. This imbalance in the sperm-egg ratio is attributed to the lack of gamete fusion. This evidence provides additional support for the existence of a localized attraction mechanism that directs sperm towards unfertilized eggs within the ampulla.

Several studies conducted on mammals, including humans [28,29,30], initially supported the idea of an undefined chemical attraction mechanism originating from the egg’s microenvironment [31, 32] (cumulus cells or the egg itself [10]), which could be replicated in vitro using egg-conditioned media [28]. Early research focused on the follicular fluids’ ability to attract sperm, revealing that follicular fluids could induce sperm accumulation in approximately 50% of cases in vitro [33]. Subsequent studies in mice and rabbits used a chemotactic Zigmond chamber [34], designed to establish chemical gradients, to test if motile sperm could respond to even a 1% concentration change of putative chemo-attractant candidates [34]. These experiments identified subpopulations of sperm in mice and rabbits that reacted to follicular or oviductal fluids [29, 30], showing significantly enhanced sperm motility parameters such as curvilinear velocity, total distance traveled per unit time, linear velocity, and linearity [29, 30].

More studies in mice and humans demonstrated that ovulated eggs or cumulus cells could also attract sperm in vitro. Crude extracts from mouse ovulated eggs induced significant changes in sperm dynamic parameters, including curvilinear velocity, linear velocity, linearity, progressive motility, and directionality [32]. In humans, using the Zigmond chamber, media conditioned with MII human eggs or their surrounding cumulus cells attracted around 8% of human sperm [28]. These findings align with previous reports showing a low number of human sperm responsive to chemo-attractive stimuli [35,36,37,38].

Unfortunately, no genetic follow-up studies have confirmed the existence of signaling mechanisms mediating attraction to unfertilized eggs (or repulsion from fertilized ones), leaving their identity and role in fertilization still unknown. One could hypothesize the presence of a molecule responsible for this short-distance attraction; its absence would result in a loss of sperm attraction to the egg, potentially leading to female infertility (sperm unable to locate eggs in the ampulla) or severe subfertility (sperm may randomly find a few eggs but miss the majority).

In addition, a few studies suggest that a gradient of progesterone, ranging from pico- to micro- molar concentrations, extend from the periphery of the cumulus mass towards the ovulated egg and facilitates the attraction of sperm towards unfertilized eggs by activating the sperm-specific flagellar and Ca2+-selective channel CatSper [39,40,41]. This model would raise the prediction that all Cd9Null eggs should be located and crossed by sperm within the ampulla. However, our observations revealed that only a minority of Cd9Null oocytes showed sperm within the perivitelline space, suggesting that a progesterone gradient may not be sufficient to ensure effective sperm attraction and distribution to all oocytes within the ampulla.

Another hypothesis posits that the observed 1:1 sperm-egg ratio may not be influenced by a chemotactic gradient, but, rather, by a post-fertilization interference to sperm binding capacity to the zona pellucida, which would occur hours after fertilization [42, 43]. Upon binding to the intact ZP2 protein in the zona [44, 45], sperm cross the zona and fuse with the oolemma. Following fusion, cortical granules release ovastacin, a metallo-endoprotease that cleaves ZP2 [46], rendering the zona inaccessible to further sperm binding (complete block to sperm binding [47]). Thus, sperm encountering a fertilized egg with cleaved ZP2 would move on to find an unfertilized egg. Although it is plausible that such a mechanism may be sufficient to ensure a 1:1 sperm-egg ratio in the ampulla, it may not be as effective, as complete block to sperm binding typically takes hours after gamete fusion [47]. In this instance, the remaining unbound sperm would continue to be active and move throughout the ampulla attempting to find eggs randomly, including fertilized eggs that have already achieved gamete fusion.

By identifying and utilizing molecules mediating sperm attraction (proven necessary in gene-edited animals), it will be possible to improve sperm selection methods. Instead of solely relying on morphological features [48], embryologists could select sperm based on their ability to be attracted to unfertilized eggs. This approach could be especially beneficial during intracytoplasmic sperm injection (ICSI), where a single sperm is directly injected into an egg. By gaining deeper insights into sperm functionality, we could potentially enhance the chances of successful pregnancies and healthy births of embryos conceived by ICSI.

Conclusions

The findings of this study indicate that the genetic disruption of gamete fusion affects the typical 1:1 sperm-egg ratio. This highlights the role of gamete fusion in maintaining this even ratio across the majority of oocytes within the ampulla. Future research will explore molecules released by unfertilized eggs that potentially facilitate attraction, as well as molecules from fertilized eggs that may induce sperm repulsion or encourage redirection to unfertilized eggs.

Data availability

All the data shown in this manuscript are included within the article.

Abbreviations

ANOVA:

Analysis of variance

DIC:

Differential interference contrast

eCG:

Equine chorionic gonadotropin

hCG:

Human chorionic gonadotropin

HSD:

Honestly significant difference

IACUC:

Institutional animal care and use committee

KO:

Knockout

LSM:

Laser scanning microscope

MII:

Metaphase II

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

s.e.m:

Standard error of the mean

TU:

University of tulsa

WGA:

Wheat germ agglutinin

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Acknowledgements

We thank Drs. Mark Buchheim, Estelle Levetin, Mrs. Marsha Howard and Lilian Ghanem at the University of Tulsa for their generous support and help with animal husbandry. The Cd9 null line was generously provided by Dr. Jurrien Dean at the National Institutes of Health (Bethesda, USA).

Funding

This study was supported by the Sidra Medicine Grant (SDR400185) to MA, the University of Tulsa, Department of Biological Science, Office of Research and Sponsored Programs (Faculty Research Grant and Faculty Research Summer Fellowship) to MA, and by the University of Tulsa, the Tulsa Undergraduate Research Challenge (TURC) program to ES.

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Authors and Affiliations

  1. Department of Biological Science, University of Tulsa, Tulsa, Oklahoma, USA

    Eva Stickler & Matteo Avella

  2. Research Branch, Sidra Medicine, Doha, Qatar

    Matteo Avella

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Contributions

ES and MA designed the study. ES performed mating experiments. ES and MA carried out sample collections, imaging and data analyses and interpretation. MA and ES wrote the manuscript.

Corresponding author

Correspondence to Matteo Avella.

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Ethics approval and IACUC Protocols

Experiments involving normal or Cd9Null mutant mice were performed in compliance with the guidelines of the Animal Care and Use Committee of the University of Tulsa, following approved protocols TU-0050 and TU-0050R1.

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The authors declare no competing interests.

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Stickler, E., Avella, M. Disruption of gamete fusion alters the sperm-egg ratio at gamete interaction. J Transl Med 22, 1134 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-024-05948-4

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-024-05948-4

Journal of Translational Medicine

ISSN: 1479-5876

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