Proceedings of the workshop "sperm-mediated gene transfer: advances in sperm cell research and applications." Siena, Italy May 23-6, 1999. Conclusions
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Journal Article↗
Biomedical subjects
B Baccetti
Publications and source records attributed to B Baccetti.
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.
CD4 Antigens↗
Embryo development, pregnancy and twin delivery after microinjection of 'stump' spermatozoa.
Intracytoplasmic sperm injection was performed with immotile spermatozoa affected by tail 'stump' defect, and resulted in normal fertilization, embryo transfer and pregnancy in a 35-year-old female. The husband had a consanguineous ancestry. Two healthy babies, a male and a female, were born and this confirms that male infertility due to certain genetic sperm defects can be overcome by the intracytoplasmic sperm injection-assisted reproduction technique. The likely genetic origin of this sperm defect and the probability of the male offspring inheriting this sperm defect should be considered. The fertilization ability of stump spermatozoa, microinjected into the oocyte, is explained on the basis of experience from our previous research.
Adult↗
A quick molecular method for the simultaneous detection in spermatozoa of nuclear, acrosomal and axonemal structure by fluorescent microscopy.
Ejaculated spermatozoa from infertile men presenting to our laboratory for semen analysis were processed with a new molecular method which reveals simultaneously, in the same sperm cell, the status of the acrosome, by testing the hyaluronidase content, the texture of the nucleus, by checking the DNA strands breaks, and the structure of the axoneme, revealing the tubulin content. The presence of hyaluronidase and tubulin is essential for the sperm function, and the analysis of the DNA status reveals the eventual apoptotic process. Using this method in normal spermatozoa, the staining of the acrosomal hyaluronidase reveals, by yellow-green fluorescence, the shape of the acrosomal complex and its texture. At the same time, in the same sperm cell, the staining of the axonemal tubulin demonstrates, by a red labeling, the presence of the protein and therefore the consistence of the axonemal structure. Simultaneously, at the head level, the absence of red labeling from nuclear DNA indicates that the apoptotic process is not present. This protocol allows quantification of the frequency of the presence of normal or abnormal spermatozoa, by an easy scoring and calculation of the apoptotic sperm or of the sperm with generic defects at acrosomal or flagellar level. The percentage of normal spermatozoa evaluated by the triple staining method has been compared with the results of the PAP staining and of the ultrastructural analysis, statistically elaborated. Triple staining results more severe than the PAP method, but TEM analysis is the finest technique to detect sperm abnormality because it considers the entire panel of sperm defects.
Acrosome↗
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.
Animals↗
Sperm/DNA interaction: integration of foreign DNA sequences in the mouse sperm genome.
Foreign DNA is spontaneously taken up by mouse epididymal sperm cells and is further internalized into nuclei. The interaction and/or internalization of the exogenous DNA triggers the activation of sperm endogenous nucleases which mediate rearrangements of the internalized DNA. Foreign DNA sequences are found to be tightly bound to the sperm nuclear scaffold, and to undergo a recombination process with the sperm chromosomal DNA. Sequence analysis of randomly selected clones from a library of sperm genomic DNA transformed with pSV2CAT plasmid showed that foreign sequences were integrated in a unique site of the sperm genome. Preliminary results suggest that the integration process is mediated by a retrotranscription step.
Animals↗
Detrimental effects of polyvinylpyrrolidone on the ultrastructure of spermatozoa (Notulae seminologicae 13).
The effect of in-vitro treatment by polyvinylpyrrolidone (PVP) on the ultrastructure of human spermatozoa has been tested previously with the statistical analysis of B. Baccetti et al. (1995, J. Androl., 16, 356-371). PVP had a primary detrimental action on the plasma membrane, as well as on acrosomal and mitochondrial membranes. Furthermore, membrane damage induces deterioration of the chromatin, axonemal tubules, fibrous sheath, and accessory fibres.
Acrosome↗
Localization of human follicle-stimulating hormone in the testis.
Localization of the follicle-stimulating hormone (FSH) molecule and its receptor (FSHR), as well as the role of FSH in Sertoli cell mitosis and maturation, has been demonstrated by several investigators in human and murine testis by detecting the localization of anti-FSH antibodies or [(131)I]-labeled FSH and by detecting FSH receptor (FSHR) mRNA by in situ hybridization, or FSHR by anti-FSHR antibodies. The presence of FSH in germinal cells is controversial or, in humans, excluded. We have investigated the distribution of the human FSH molecule and its receptor in human and mouse testicular cells under different experimental conditions, at the submicroscopical level, by using a better antigenicity conservative procedure. Thus, the distribution of FSH and of the messenger RNA for its receptor in Sertoli cells has now been clarified. In germinal cells, our observations demonstrate the presence of FSH and the FSHR mRNA: the first on the plasma membrane and in endocytotic vesicles, and the second scattered in the cytoplasm. The cells presenting the higher amount of positivity ranged from spermatogonia to spermatocytes, including round spermatids. Penetration was by the endocytosis via membrane vesicles in which the FSHR is present, whereas its messenger is largely present in the cytoplasm and is responsible for the binding and subsequent internalization of the FSH molecule. As a control, human FSH was administered in vitro to the Y1 mouse cell line, which was stably transfected with cDNA for FSHR and devoid of endogenous FSH. The FSH molecule has been localized by monoclonal antibodies on plasma membranes and vesicles, and the FSHR mRNA was found scattered in the cytoplasm after in situ hybridization. We can now conclude that FSH is present in Sertoli cells and in round germinal cells, both expressing the FSHR. FSH penetrates in a similar way in both kinds of cells via endocytosis, and is therefore subsequently localized in the same membranous organelles.
Animals↗
Localization of lamins in mammalian spermatozoa.
Little information is available concerning lamins in the nucleus of germinal cells. In this paper we briefly describe and compare the organization of A- and B-type lamins in several mammalian spermatozoa. Nuclear lamin B is localized primarily in the postacrosomal sheath of human, bull and rabbit spermatozoa; lamin A/C is a major component of the equatorial segment in most of mammalian sperm, with the exception of rodents. In mouse sperm, devoid of equatorial segment, only lamin B appears to be expressed. The same happens in human pathological spermatozoa in which the equatorial segment is altered or absent.
Animals↗
G protein-coupled receptor kinase GRK4. Molecular analysis of the four isoforms and ultrastructural localization in spermatozoa and germinal cells.
G protein-coupled receptor kinase 4 (GRK4) presents some peculiar characteristics that make it a unique member within the GRK multigene family. For example, this is the only GRK for which four splice variants (GRK4alpha, -beta, -gamma, -delta) have been identified. We developed a simple assay to study kinase activity, and we found that GRK4alpha, but not GRK4beta, -gamma, and -delta, was able to phosphorylate rhodopsin in an agonist-dependent manner. GRK4alpha kinase activity was inhibited by Ca2+/calmodulin (CaM) (IC50 = 80 nM), and a direct interaction between GRK4alpha and Ca2+/CaM was revealed using CaM-conjugated Sepharose 4B. The other three GRK4 isoforms did not interact with CaM in parallel experiments. The present investigation also aimed to define cellular and ultrastructural localization of GRK4. A substantial expression of GRK4 mRNA was only found in testis and in the spermatogonia cell line GC-1 spg. Specific GRK4 immunoreactivity was only found on sperm membranes, and immunochemical and ultrastructural analyses showed that it is associated to the acrosomal membranes and to the outer mitochondrial membranes. GRK4gamma was the only detectable isoform in human sperm. We concluded that: i) only GRK4alpha can phosphorylate rhodopsin and that this activity is inhibited by CaM; ii) the other three isoforms do not phosphorylate rhodopsin and do not interact with CaM; and iii) the association of GRK4 with highly specialized sperm organelles, which are essential for fertilization, strongly indicates that this kinase is involved in this process.
Animals↗
The effect of follicle stimulating hormone therapy on human sperm structure (Notulae seminologicae 11).
The effects of follicle stimulating hormone (FSH) treatment on the quality of human spermatozoa were assessed by examining the ultrastructure and the function of infertile human spermatozoa using a previously-defined formula. Using the spermatozoa as an andrological monitor shows that the therapeutic effect of FSH depends on the type of sperm defect. The response to FSH is, in many cases, positive and can be evaluated by examining the state of the ejaculated spermatozoa. From an initial group of 81 patients, 15 were placebo-treated controls, and 19 were non-responders (mainly with microbially infected semen). Out of 47 responders, after therapy nine achieved improved sperm quality which approached the natural fertility threshold. These responders all had spermatozoa affected by immaturity or apoptosis (n = 27). The 20 microbially-infected responders also had immature spermatozoa and never achieved the quality level of natural fertility. Thus, a natural fertility level was only achieved by nine responders out of 27 (three with immature spermatozoa, and six with apoptotic spermatozoa). Using our method of sperm analysis, these patients' spermatozoa were clearly categorized before treatment as either immature or apoptotic. In consequence, the success of the therapy was predictable. The response of individual organelles to therapy was examined. Certain qualities of the acrosome, the chromatin, the mitochondria, and the axoneme appear to be sensitive to FSH. Most of the previous conflicting results reported in the literature may be due to a lack of relevant discrimination between the different defects present in the spermatozoa of the patients, without assessing the likelihood of their response.
Acrosome↗
Submicroscopic mathematical evaluation of spermatozoa in assisted reproduction. 3. Partial zona dissection (PZD) (Notulae seminologicae 12).
This paper belongs to a series of application of the Baccetti's et al. (1995) formula to the submicroscopical mathematical examination of the human spermatozoa used for assisted reproduction. The present experiment concerns partial zona dissection, a technique requiring a careful evaluation of sperm quality in order to predict the success of the program. Our results demonstrate that the sperm submicroscopic characters introduced in the formula are clearly correlated with the result of PZD. In fact the two numbers concerning the sperm ultrastructural quality (percentage and total number of spermatozoa free from defects in the ejaculate) obtained in successful and unsuccessful PZD groups, showed a large difference (P < 0.01). The most important characteristics seem to be the quality of the acrosomal complex, the condition of the chromatin and the mitochondrial assembly. All these characteristics are expressed with largely different means in successful and unsuccessful ejaculates (from P < 0.05 to P < 0.01). A comparison with the results previously obtained in ICSI (Strehler et al., 1995) and IVF (Piomboni et al., 1996) shows that sperm quality is significantly more implicated in the success of IVF than of PZD or ICSI.
Female↗
Submicroscopic mathematical evaluation of spermatozoa in assisted reproduction. 4. The bovine fertilization (Notulae seminologicae 10).
In this paper we apply a modification of the formula of Baccetti et al. (1995) in the evaluation of submicroscopical characteristics of bull spermatozoa used in assisted reproduction. In the present experiment sperm quality is proposed as a useful parameter in predicting the success of fertilization. Our results demonstrate that the percentage of spermatozoa devoid of submicroscopic defects, according to the particular Bayesan formula proposed by us, is clearly correlated with the result of artificial insemination. In fact, the parameter concerning sperm quality obtained in variously successful donors shows a large correlation with fertility power. The synthetic parameters observed are therefore a good tool in the prediction of sperm power in artificial fertilization. The evaluation is mainly concerned with the quality of the acrosomal characters, the status of the chromatin, the shape of mitochondria, the position of the postacrosomal sheath, the perinuclear space and the axonemal pattern. All these characters are expressed with different means in ejaculates. All these data confirm that submicroscopic-mathematical evaluation offers a convincing and reliable diagnosis based upon sperm structure and functions such as acrosomal reaction and cell motility. It has been also demonstrated that sperm quality is a major factor in the success of artificial insemination and it is clearly revealed in the integrity of most of sperm organelles.
Animals↗
Submicroscopic mathematical evaluation of spermatozoa in assisted reproduction. 2. In vitro fertilization. (Notulae seminologicae. 7)
This paper belongs to a series of applications of the Baccetti et al. formula (1) to the submicroscopical mathematical examination of human spermatozoa used for assisted reproduction. The present experiment concerns IVF, a technique requiring careful evaluation of sperm quality to predict the success of the program. Our results demonstrate that the sperm submicroscopic characters introduced in the formula are clearly correlated with the result of IVF. In fact the two numbers concerning sperm quality (percentage of spermatozoa free from structural defects and total number in the ejaculate of spermatozoa free from defects) obtained in successful and unsuccessful IVF groups, showed a large difference. The t distribution in both cases reached a significance of 0.005. The synthetic parameters obtained are therefore a good tool in the prediction of sperm power in in vitro insemination techniques. The most important characteristics seem to be the quality of the acrosomal complex, the status of the chromatin, the shape of mitochondria, the axonemal pattern, and the membrane integrity. All these characteristics are expressed with largely different means in successful and unsuccessful ejaculates (t distribution significant at 0.005). All these data confirm that submicroscopic mathematical diagnosis offers a convincing evaluation of sperm structure and function, involving all organelles, including acrosome function and cell motility. It is also demonstrated that sperm quality is a major factor in the success of IVF and that it is clearly revealed by the integrity of the majority of the sperm organelles.
Acrosome↗
Apoptosis in human ejaculated sperm cells (notulae seminologicae 9).
In this work we apply the Hoechst 33258 DNA staining, the TUNEL procedure and conventional electron microscopy to study the ejaculate of fertile and infertile men, in order to detect apoptosis in human sperm cells. We have observed that apoptosis is abnormally frequent in the sperm cells of the ejaculate of sterile men, and that it shows the classical biochemical and ultrastructural pattern in spermatozoa, spermatids and apoptotic bodies. These characteristics, involving the chromatin, the nuclear envelope, the plasma membrane, the presence of cytoplasmic vacuoles and the status of mitochondria, are consistent whatever the pathology of the patient is. What is varying is the percentage of the apoptotic sperm cells, that is about 0.1% in fertile controls, and increases up to about 10% in varicocele, infected (including AIDS), "round headed' patients, to 20% in cryptorchid men, to 25% in immature patients, and to 50% in testicular seminoma carriers. Obviously in each category the frequence of apoptotic cells increases concomitantly with the degree of the affection. The present demonstration not only extends to human spermiogenetic epithelium the natural presence of apoptosis, which starts in the testis and is revealed in the ejaculate, but also explains many abnormal ultrastructural sperum patterns hitherto unexplained in fertile and infertile individuals.
Apoptosis↗
Localisation of two classes of acetylcholine receptor-like molecules in sperms of different animal species.
The distribution of different classes of acetylcholine (ACh) receptor-like molecules in sperms of different invertebrate and vertebrate species is described. ACh receptor molecules belong to one of two classes: muscarinic receptors (mAChRs), associated with signal transduction mechanisms in the inner domain of the cell, and nicotinic receptors (nAChRs), capable of opening Na+ channels when activated by the ligand. Molecules immunologically related to mAChRs and to ACh can be identified by specific antibodies, and revealed by immunofluorescent or immunogold staining; the nicotinic receptor-like molecules are localised as curare-sensitive affinity sites for alpha-bungarotoxin. In all species studied, both classes of receptors were found, with a similar distribution. Muscarinic-like molecules were found mainly in the sperm head regions of most species; such a localisation may be correlated to a function in sperm-egg interaction, for instance in the regulation of the block to polyspermy. Nicotinic-like molecules are present mainly in the tail and in the post-acrosomal region of most animals, thus confirming their function in the regulation of sperm propulsion, but are also present at the acrosomal region of most species. The distribution patterns of the different classes of molecules indicate that both may be involved in sperm-egg interactions, in addition to their known function in the regulation of sperm propulsion.
Acrosome↗
Submicroscopic mathematical evaluation of spermatozoa in assisted reproduction. I. Intracytoplasmic sperm injection. (Notulae seminologicae 6).
After a large introduction concerning the influence of sperm quality in the success of in vitro fertilization, the debated problem of the importance of the sperm quality in intracytoplasmic sperm injection is investigated. The spermatozoa are studied by electron microscopy, and the results evaluated by the formula of Baccetti et al. (1995). The quality of spermatozoa has been correlated with the success of ICSI. The first conclusion has been that ejaculates which produced embryos contained higher percentages and overall higher total number of 'healthy' spermatozoa than those which obtained fertilizations arrested at the two pronuclei stage, or showed no fecundation at all. A second conclusion was that the quality of sperm organelles is mainly involved in the oocyte activation, but after the 2 pronuclei stage, segmentation usually proceeds with few influences from sperm quality. The highest involvement of spermatozoa is therefore the initiation of oocyte activation. A third conclusion confirmed the need of ultrastructural evaluation of sperm cell organization, deeply investigating the inner organelles. Moreover, we observed that the head organelles are more involved that the tail ones. Three acrosomal characteristics are concerned: the shape, the dimensions and the content. All of them are significantly better in the oocyte activating than in non activating spermatozoa. Also the nuclear shape and the status of the chromatin (frequently strictly interdependent) are significantly concerned with the oocyte activation and with the embryo segmentation. In the tail, the mitochondrial shape is significantly different in activating and not activating spermatozoa, and also in those producing embryo segmentation. Moreover, the absence of dynein arms seems to be determinant in impeding the oocyte activation.
Fertilization in Vitro↗
HIV-particles in spermatozoa of patients with AIDS and their transfer into the oocyte.
By immunocytochemistry and in situ hybridization at the electron microscopy level, and by the PCR technique, we have shown that HIV-1 binds and enters normal sperm; that viral particles, their antigens, and nucleic acid are present in sperm from HIV-1 infected men; and that such sperm can transfer HIV-1 like particles to normal human oocytes. We also present evidence that a galactosylceramide-like compound is present on the sperm membrane and could function as an alternative receptor for HIV.
Acquired Immunodeficiency Syndrome↗