Human Gametes Research Articles

Human Sperm-Oocyte Recognition and Infertility

The incidence of infertility related to both male and female factors continues to rise despite many advances in reproductive technologies. Some abnormalities in human gamete interaction have been shown to be due to defects in the sperm, and others have been attributed to defects in the zona pellucida (ZP). Our lack of understanding of molecular mechanisms involved in the interaction of human sperm with the ZP in fertile as compared with infertile females and males has been limited because of the unavailability of human oocytes and ethical restraints on experimental studies. It is becoming increasingly apparent that improved clinical assays are necessary for evaluating sperm-ZP interaction in order to assess the optimal procedures for successful fertilization and pregnancy. With advances in molecular biology, the genes encoding the three major human ZP proteins have been identified and complementary DNAs are available to begin to better evaluate the molecular basis of sperm-ZP interaction.

Cryopreservation of the human female gamete: current and future issues.

pregnancy and live birth (Tucker et al., 1998b). The potential advantages associated with the ability to freeze Two important issues, however, remain to be resolved before and store human oocytes successfully have been well estabthe role of this technology in current practice can be fully lished for some time. Not only would it allow the circumvention elucidated. Firstly, it would be preferable if continuing research of moral, ethical and legal problems which arise as an inevitable could determine whether modifications to the above method consequence of embryo freezing, but also it would offer the could improve outcome. A tendency to adopt techniques widely prospect of broadening the fertility options available to women when they show promise often occurs at the expense of further who, for a variety of medical reasons, are likely to lose ovarian refinement. For example, a recent study has suggested that function prematurely. Banks of frozen donated oocytes would optimal survival of human oocytes may be achieved at an icefacilitate the donation process, which is often complicated by seeding temperature which is higher than that used in current a requirement for donor–recipient synchrony. More controclinical oocyte cryopreservation protocols (Trad et al., 1998). versially, oocyte storage would also open the door to the This is particularly important when we consider the second possibility of women, with no medical indications and no issue, which concerns the relative efficiency of oocyte and immediate plans to conceive, being able to store ‘young eggs’ embryo freezing. If we are to consider mature oocyte freezing for potential use at a later date. as an alternative to embryo freezing then we need to be aware Technically, the possibility of offering oocyte cryopreservof the likely return from the available material in each case. ation as a routine procedure had seemed somewhat remote In other words we need to ask ‘How many implantations can after failure to reproduce early reports of success with a we expect per 100 fresh oocytes available?’. Figures from our dimethylsulphoxide-based method in the 1980s (Chen, 1986; own unit suggest that ~90% of collected oocytes may be Al-Hasani et al., 1987; Van Uem et al., 1987). The difficulties expected to be mature, that ICSI would be expected to result encountered in achieving success with human mature (metain normal (dipronucleate) fertilization in ~60% of injected phase II) oocytes were postulated to arise mainly from inherent mature oocytes, and that ~90% of early cleavage stage (day problems associated with the susceptibility of the mammalian 2) embryos generated would be considered suitable for cryospindle to freezing induced damage. Such problems were preservation. This means that just under 50 embryos would highlighted by reports of elevated rates of post-thaw aneuploidy be frozen per 100 oocytes collected in a unit where embryo in cryopreserved mouse oocytes (Kola et al., 1988). However, utilization/cryopreservation rates are high. Our post-thaw work by our group using propanediol and sucrose as cryoresults suggest that, although only 15% of all embryos frozen protectants in conjunction with a slow freeze/rapid thaw fail to survive cryopreservation, only 55% of frozen embryos method based on that successfully used for post-fertilization will survive with all blastomeres intact after thawing. The stages in the human, suggested that this pessimism was not percentage of fully intact thawed embryos which develop to justified. We showed that oocytes could survive cryopreservthe fetal heart (FH) stage following transfer in our unit is ation without compromising spindle integrity (Gook et al., ~12%, a figure which is almost double the value for embryos 1993), that post-thaw fertilization was characterized by normal which have suffered blastomere loss as a result of cryopreservkaryotypes and an absence of stray chromosomes (Gook ation. Taking all the above into account would result in an et al., 1994), and that frozen/thawed oocytes fertilized by

The brave new world of making babies

Following the breakthrough of in-vitro fertilization (IVF) after half a century of vigorous investigation initial criticism ranged from skepticism to sheer outrage. However IVF was successfully introduced throughout the world in the early 1980s. Since then complex and expensive ovarian stimulation protocols have appeared to obtain large numbers of oocytes for fertilization in vitro. Presently the total number of IVF is approaching half a million worldwide with an estimated average pregnancy rate of 10-15% per embryo transfer. Focus of the study has changed from empirical endocrine and surgical approaches for fertility therapy to IVF. Since IVF many development have emerged which further raises ethical issues. With these developments it has created entirely new possibilities for future procreation plus highly complex questions about ethical legal psychosocial and medical boundaries. Fertility specialists and biologists now have gametes under their controls enabling procreation with desired genes at a desired time and in the desired female. However the manipulation of human gametes or embryos may induce subtle but far-reaching changes in the genetic material of an offspring. Therefore it is still best to rely on the doctors opinion.

New aspects of cryopreservation of oocytes and embryos in assisted reproduction and future perspectives.

Cryopreservation of human gametes and tissue has been introduced into assisted reproduction in recent years. The current status as well as new developments of cryopreservation, especially in conjunction with new techniques, are reviewed. Cryopreservation of pronuclear stage oocytes or embryos at the cleavage stage are routine procedures worldwide after conventional in-vitro fertilization (IVF). The data on children born show no increase in malformation rates, or any impaired psychomotor development. The combination of cryopreservation with intracytoplasmic sperm injection (ICSI) has given the same results. Cryopreservation of unfertilized mature and immature oocytes has lead only to a few pregnancies until now. It is still unknown whether ICSI may help to improve these results. Experience using cryopreservation of blastocysts is still limited, with a wide range of pregnancy rates (0-53 %) being reported. Further studies are required before exact numbers for these procedures can be given. The combination of biopsy and cryopreservation appears to have a different impact for subsequent embryo development in animals and humans. Possibly, there is a reduction in embryo developmental potential after these procedures. Currently, the most promising technique is the cryopreservation of ovarian tissue, which has already led to the birth of lambs, mice and rats. The future development of cryopreservation is discussed.

Failure of HIV-1 to Infect Human Oocytes Directly

In this study, the susceptibility of mature human oocytes to HIV-1 infection has been investigated. We exposed in vitro human oocytes of healthy women using inocula of cell-free HIV-1. We also tested for the presence of HIV-1-specific receptor molecules on the surface of these cells. By applying polymerase chain reaction (PCR) analysis, transmission electron microscopy (TEM), and immunocytochemistry at both light and electron microscopic levels, we did not obtain evidence for HIV DNA production nor for oocyte-associated HIV particles. Experiments of immunostaining for CD4, CCR5, and GalAAG (putative receptor for HIV in sperm), as well as reverse transcriptase (RT)-PCR for CD4, CCR5, and CXCR4, which all suggested the absence of the mentioned receptors in mature oocytes and in follicular cells. This study fills an important gap concerning the information available on the direct HIV infection of human gametes, adds to our basic understanding of HIV infection in human oocytes, provides different results from those obtained with human spermatozoa using comparable methods, and provides a basic contribution to the investigation on HIV infection in human oocytes.

Failure of HIV-1 to Infect Human Oocytes Directly

Summary: In this study, the susceptibility of mature human oocytes to HIV-1 infection has been investigated. We exposed in vitro human oocytes of healthy women using inocula of cell-free HIV-1. We also tested for the presence of HIV-1-specific receptor molecules on the surface of these cells. By applying polymerase chain reaction (PCR) analysis, transmission electron microscopy (TEM), and immunocytochemistry at both light and electron microscopic levels, we did not obtain evidence for HIV DNA production nor for oocyte-associated HIV particles. Experiments of immunostaining for CD4, CCR5, and GalAAG (putative receptor for HIV in sperm), as well as reverse transcriptase (RT)-PCR for CD4, CCR5, and CXCR4, which all suggested the absence of the mentioned receptors in mature oocytes and in follicular cells. This study fills an important gap concerning the information available on the direct HIV infection of human gametes, adds to our basic understanding of HIV infection in human oocytes, provides different results from those obtained with human spermatozoa using comparable methods, and provides a basic contribution to the investigation on HIV infection in human oocytes.

Equality and selection for existence.

It is argued that the policy of excluding from further life some human gametes and pre-embryos as "unfit" for existence is not at odds with a defensible idea of human...

Expression of interleukin-1 system genes in human gametes.

There is considerable evidence that the interleukin-1 (IL-1) system plays an important role in ovarian and testicular physiology, implantation, and other reproductive events. Human embryos express IL-1beta, IL-1 receptor type I (IL-1RtI), and IL-1 receptor antagonist (IL-1RA) at both the mRNA and protein levels. The presence of IL-1alpha and IL-1beta in oocyte-conditioned media and on the surface of human oocytes suggests that these cells may also produce this cytokine; however, whether the IL-1 system gene products are present as stable mRNAs in human gametes (oocytes and spermatozoa) has not yet been demonstrated. We used stringent cell separation techniques combined with reverse transcription-polymerase chain reaction to investigate the expression of various IL-1 system genes (IL-1alpha, IL-1beta, IL-1RtI, and IL-1RA) in human gametes and cumulus cells. Our results indicate that freshly isolated cumulus cells express all these IL-1 system components. On the other hand, IL-1alpha, IL-1beta, and IL-1RtI mRNAs were not found in either unfertilized or fertilized human oocytes, and a very few metaphase II human oocytes had transcripts for either secreted (10%) or intracellular (17%) IL-1RA. Mature spermatozoa did not contain mRNA for any of the of the IL-1 system components. The absence of informational RNA for the IL-1 system components in human unfertilized and polyploid oocytes and fresh immature oocytes suggests that maternal transcripts for these genes do not contribute to early embryo development. The presence of IL-1 components at the protein level in human oocytes may be due to binding of IL-1 produced by cumulus cells or other cell types, or to prior intrafollicle transcription and translation. Likewise, IL-1 system components do not appear to have a physiological role in mature spermatozoa since none of these components are present at the mRNA or protein levels, and important functional parameters such as motility and acrosome reaction appear not to be affected by IL-1beta in vitro. However, the abundant expression of IL-1alpha, IL-1beta, the IL-1RtI, and its antagonist IL-1RA by human cumulus cells provides further evidence that the IL-1 system plays a role in human ovarian physiology.

The origin, effects and control of air pollution in laboratories used for human embryo culture.

Testing shows that most laboratories conducting human gamete and embryo culture have air quality and sources of contamination that exceed the levels measured in homes, businesses and schools. The sources of these contaminants have been shown to be either from activities outside the laboratory, or emitted from materials used in the facility, such as compressed gas, cleaning and sterilizing agents, plastic and stored materials. Both the laboratory structure and the air handling systems may affect the air composition. The significance of these findings is being validated by the accumulation of field case studies and now by assay procedures. Products given off by road sealant were shown to have accumulated in one of the examined laboratories, adjacent to a large re-surfaced parking area. Aldehydes such as acrolein, hexanal, decanal, pentanal and others were detected at elevated concentrations that were statistically significant. Since it is not appropriate to add potentially suspect chemicals to human embryos, we used a mouse-model to study the effect of acrolein. The growth of mouse embryos was significantly affected after acrolein was added at different concentrations to the culture environment. The physiological effect was noted at concentrations in the low ppm range. The testing end-point of embryo death must still be considered to be a crude basis for evaluating toxicological effects, since it involves addition of compounds to culture media and unprotected growth until the blastocyst stage. The findings may, however, support observations of decreased pregnancy rate following exposure of human embryos to aldehydes or other adverse conditions. With proper engineering and material selection, it is possible to reduce such contamination. The usefulness of this approach for controlling aldehydes has been demonstrated by decreasing levels in the laboratory to below those of the outside air.

The debate on the presence of HIV-1 in human gametes

The debate about the presence of HIV-1 particles in human gametes and recent experimental results are reported in detail. Using immunocytochemistry, in situ hybridization at electron microscopy level, polymerase chain reaction and in vitro fertilization, it has been demonstrated that human spermatozoa can incorporate HIV-1 using special receptors, different from the usual CD4, and that they remain active and able to vehicle the viral particles into the oocyte, which is regularly fertilized. Moreover, by transmission electron microscopy (TEM), immunocytochemistry and PCR, we demonstrated that cell-free HIV-1 is not able to bind and penetrate the human oocyte in vitro. We attribute this behaviour to the fact that the oocyte and cumulus cells are devoid both of GalAAG and of CD4 receptors. PCR analysis indicated that mRNAs specific for CD4, CXCR4 and CCR5 proteins were absent, too.

Long PCR Analysis of Human Gamete mtDNA Suggests Defective Mitochondrial Maintenance in Spermatozoa and Supports the Bottleneck Theory for Oocytes

The long PCR and the Southern blot techniques were used to study mitochondrial DNA (mtDNA) in 94 sperm samples, and in 35 oocytes collected from 12 women. The sperm samples were classified in two sets: 37 samples from normal subjects, and 57 samples from patients with oligoasthenospermia. In both sets, most of the spermatozoan mitochondria had multiple mtDNA deletions. The rate of mtDNA mutation, which appears unexpectedly high, considering the short life span of the spermatozoa, may be due to impaired maintenance during differentiation. In contrast, despite the long life span of oocytes and the extended meiotic period, oocyte mitochondria showed few mtDNA rearrangements. However, mitochondria in oocytes from a given donor revealed considerable mutational heterogeneity. This supports the bottleneck theory of rapid segregation of mtDNA genotypes during early oogenesis. The long PCR technique, which allows analysis of the entire mitochondrial genome, provides new information on mtDNA instability in human gametes. Our findings suggest that mtDNA maintenance differs in the two types of gametes.

An update on human fertilization.

The process of fertilization and the role that each gamete plays in that process have been the subject of investigation in a large number of species and for many years. However, while much is now known for some species relatively little is known for others. Indeed, the specific events that are required to occur in the human male and female gametes and that facilitate fertilization are still somewhat ill-defined. For example, as of today, there have been numerous biomolecular processes that have been put forth as playing an important role in the sequence of events during which sperm acquire fertilizing ability, yet the actual significance of many of these remains suspect. This article will summarize what is presently best known about prefertilization processes occurring in human spermatozoa. For those interested in nonhuman mammalian and nonmammalian species, articles addressing this and other topics can be found in the many review articles cited herein.

HLA expression on immature and mature human germ cells

Human leukocyte antigens (HLA) are membrane-bound glycoproteins encoded by the human major histocompatibility complex located on chromosome 6. They are known to function in immunologic recognition and, with regard to reproduction, a number of non-immune functions have been proposed. Although the expression patterns of the major histocompatibility antigens have been extensively studied at the maternal–fetal interface, there are still controversial reports on the expression of these molecules by human gametes and preimplantation stages. This brief review focuses on recent studies where the expression and distribution of HLA on human spermatogenic cells (spermatogonia, primary and secondary spermatocytes, spermatids, spermatozoa), primary and secondary oocytes, and preimplantation embryos have been investigated. These results, and their possible implications for the fertilization process and further embryonic development, will be presented.

Glycodelins

Glycodelins are 28 to 30-kD glycoproteins synthesized in various glands, notably those of the male and female reproductive organs. Depending on the site of origin, the same protein backbone is glycosylated in different ways, yielding glycodelins with different biological actions. Thus, human endometrium-derived glycodelin-A is temporally expressed in the latter half of the menstrual cycle, consists of unique sialylated and fucosylated lacdiNAc oligosaccharide sequences, and inhibits sperm-egg binding. By contrast, glycodelin-S from seminal vesicles has no such oligosaccharide sequences and no contraceptive activity. Glycodelin-A also has potent immunosuppressive properties, and its chemically modified forms inhibit transmission of HIV in vitro. Studies are reviewed suggesting that glycoforms dictate the function of human glycodelins, and that some of the oligosaccharide recognition sites present in the human gametes and immune cells have converged.

The polypeptide backbone of recombinant human zona pellucida glycoprotein-3 initiates acrosomal exocytosis in human spermatozoa in vitro.

Human gamete interaction is of fundamental biological importance, yet the molecular interactions between spermatozoa and the zona pellucida are poorly understood. Surprisingly, the role of the polypeptide backbone of zona pellucida glycoprotein 3 (ZP3), the putative ligand for spermatozoa activation, has been largely overlooked. Purified recombinant human ZP3 was expressed in Escherichia coli as a C-terminal fusion to the dimeric glutathione S-transferase (GST) from Schistosoma japonicum and was shown to induce acrosomal exocytosis in live, capacitated human spermatozoa. The level of exocytosis is comparable with that obtained using purified, glycosylated, recombinant human ZP3 [van Duin, M., Polman, J.E.M., DeBreet, I.T.M., Van Ginneken, K., Bunschoten, H., Grootenhuis, A., Brindle, J. and Aitken, R.J. (1994). Biol Reprod. 51, 607-617]. These data imply that the polypeptide chain of human ZP3 contributes to recognition of spermatozoa during acrosomal exocytosis in vitro.

Human gamete fusion can bypass beta1 integrin requirement.

Since alpha6beta1 integrin has been shown to function as a sperm adhesion receptor in the mouse, we investigated the potential role of beta1 integrin in the gamete fusion process in humans. The expression of beta1 integrin was morphologically analysed by indirect immunofluorescence and confocal microscopy. A homogeneous and intense staining was detected at the plasma membrane, and in some subcortical vesicles of germinal vesicle stage oocytes (GV). Beta1 almost disappeared from oolemma and cytoplasm of metaphase I (MI) oocytes, but was re-expressed as asymmetrical patches at the plasma membrane of metaphase II stage oocytes (MII). A functional fusion assay based on Hoechst or calcein-AM dye transfer from one gamete to the other showed that maturing oocytes were able to fuse with an increasing number of spermatozoa (11-22 from GV to MII respectively), and that fused spermatozoa co-localized with beta1 integrin patches. Human gamete fusion was only partially inhibited either by RGD-containing peptide (GRGDTP), or by blocking anti-human beta1 integrin monoclonal antibody (DE9), with a maximum of 50% inhibition. Despite the combined addition of GRGDTP and blocking mouse anti-human beta1 integrin DE9 in the assay, a complete inhibition of fusion could not be achieved. A mouse polyclonal antibody raised against human oocyte membranes was more potent in inhibiting the fusion. Since beta1 integrin expression at the plasma membrane was not correlated to oocyte fusibility, and since it was only partially inhibited by DE9 and/or RGD peptide, we suggest that human gamete fusion can bypass the beta1 requirement. Beta1 integrin certainly participates in human gamete fusion by acting in co-operation with multiple integrin/disintegrin couples or another cofactor, not yet identified.

The polysulphate binding domain of human proacrosin/acrosin is involved in both the enzyme activation and spermatozoa-zona pellucida interaction.

Mammalian acrosin is a protease present as a zymogen in the acrosome of a non-reacted mammalian sperm, and in vitro is able to carry out limited hydrolysis of homologous and heterologous zonae pellucidae. On the other hand, sulphated polymers and zona pellucida glycoproteins bind to acrosin on a domain different from the active site, named the polysulphate binding domain (PSBD). Thus it is believed that acrosome-reacted spermatozoa bind to glycan chains of the zona pellucida through PSBD participating as secondary binding receptor. The aim of the present work was to study the role of PSBD during both human gamete interaction and acrosin activation. In this work we present evidence that the anti-human acrosin monoclonal antibody C5F10 is directed to an epitope located on or near the PSBD on human proacrosin/acrosin. Moreover, we show that this antibody is able to inhibit both proacrosin activation induced by fucoidan and the sperm binding to the zona pellucida. Our results suggest that the same PSBD is involved in both sperm secondary binding, during zona pellucida penetration, and proacrosin activation.

O-084. Fas (APO-1) receptor/Fas ligand expression in human gametes and pre-embryos in vitro

O-084. Fas (APO-1) receptor/Fas ligand expression in human gametes and pre-embryos in vitro

Expression of glycans linked to natural killer cell inhibition on the human zona pellucida.

Protection of the gametes from potential immune responses is a primary function in human reproduction. The primary cell type responsible for the innate immune response in the uterus is the natural killer (NK) cell. NK cells normally recognize Class I major histocompatibility (MHC) molecules on potential target cells. Since both human spermatozoa and human oocytes do not express Class I MHC molecules on their surfaces, the appropriate cell surface signal that abrogates potential NK cell-mediated responses directed against these gametes is unknown. Recent evidence indicates that surface expression of bisecting-type N-linked glycans protects cells sensitive to NK cell-mediated lysis. We report that the zona pellucida of the human egg and plasma membranes of human spermatozoa potentially bind a lectin probe specific for bisecting type glycans in a carbohydrate-dependent manner. Since the innate immune response in the uterus is primarily mediated by NK cells, our results indicate that human gametes may be protected from this response by expressing bisecting type N-linked glycans on their surfaces.

O-083. Programmed cell death (apoptosis) in human gametes and pre-embryos in vitro

O-083. Programmed cell death (apoptosis) in human gametes and pre-embryos in vitro