Cellular events during fertilization failure in humans(2)(2).
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Chromatin↗
Biomedical subjects
A D Vitullo
Publications and source records attributed to A D Vitullo.
Fast-developing preimplantation embryo progeny from heterogametic females in mammals.
Karyotyping and cell number estimates in preimplantation embryos from heterogametic (XY*) and homogametic (XX) females of the field mouse Akodon azarae were studied to determine whether XX-XY-XY* differences exist in the rate of preimplantation development. At the morula stage, XY embryos from heterogametic mothers had twice the mean number of cells compared with XX embryos. However, this difference in cell numbers was not seen between XX and XY embryos from homogametic mothers. In this case, mean cell numbers were similar despite embryos being XX or XY. Furthermore, the mean cell number for XX and XY morulae from homogametic females was comparable to that for XX embryos from heterogametic females. It is concluded that XY* embryos (which will develop into heterogametic females) show an accelerated rate of preimplantation development.
Animals↗
Cytoskeletal organization defects and abortive activation in human oocytes after IVF and ICSI failure.
In this study, we analysed the distribution of beta tubulins to detect spindle and cytoplasmic microtubules, alpha acetylated tubulins for sperm microtubules and chromatin configuration in oocytes showing fertilization failure after conventional IVF or intracytoplasmic sperm injection (ICSI). A total of 450 human oocytes that failed to fertilize were studied 20-40 h after IVF or ICSI. In all, 287 oocytes were stained for immunofluorescence and chromosomal spreads were performed by Tarkowski's air-drying method in 163 IVF or ICSI oocytes that did not develop pronuclei after the extrusion of a second polar body. Immunofluorescence analysis showed that the main reason of fertilization failure after IVF was no sperm penetration (55.5%). The remaining oocytes showed different abnormal patterns, e.g. oocyte activation failure (15.1%) and defects in pronuclei apposition (19.2%). On the other hand, fertilization failure after ICSI was mainly associated to incomplete oocyte activation (39.9%), and to a lesser extent with defects in pronuclei apposition (22.6%) and failure of sperm penetration (13.3%). A further 13.3% of the ICSI oocytes arrested their development at the metaphase of the first mitotic division. The chromosomal spreads allowed the analysis of abortive activations, in which no pronuclei formed but a second polar body was extruded. Immunofluorescence and cytogenetic analysis provided a useful tool to improve infertility diagnosis and prognosis in each particular case.
Chromatin↗
Successful capacitation and homologous fertilization in vitro in Calomys musculinus and Calomys laucha (Rodentia - sigmodontinae).
Small South American rodents of the genus Calomys have been used extensively for virology and ecological research. Previous studies have demonstrated that Calomys musculinus and Calomys laucha have a relatively short oestrous cycle and that superovulation and parthenogenetic activation can be induced. The purpose of this study was to determine the requirements for in vitro manipulation of the male gamete and in vitro fertilization. Two culture media and different concentrations of spermatozoa were tested for their ability to support sperm motility, hyperactivation and the acrosome reaction. The ability of capacitated Calomys spermatozoa to penetrate zona-free hamster eggs was also evaluated. In vitro fertilization was assessed by examining attachment and binding to the zona pellucida, second polar body extrusion, pronucleus formation and the fertilizing sperm tail. The results of the study showed that: (i) Tyrode's albumin lactate pyruvate (TALP) medium was more effective than T6 medium for maintaining sperm motility in vitro; (ii) hyperactivation was achieved with TALP but not with T6; (iii) the acrosome reaction was easily distinguished by light microscopy and depends on time and sperm concentration; (iv) capacitated spermatozoa are able to penetrate zona-free hamster eggs; and (v) superovulated oocytes can be fertilized in vitro. This is the first report of capacitation and in vitro fertilization for Calomys sp. These results provide opportunities to use C. musculinus and C. laucha as new laboratory animals for research into reproductive biology.
Acrosome Reaction↗
Nuclear factor I-mediated repression of the mouse mammary tumor virus promoter is abrogated by the coactivators p300/CBP and SRC-1.
To better understand the function of nuclear factor I (NFI) proteins in transcription, we have used transient transfection assays to assess transcriptional modulation by NFI proteins on the NFI-dependent mouse mammary tumor virus (MMTV) promoter. Expression of NFI-C or NFI-X, but not NFI-A or NFI-B proteins, represses glucocorticoid induction of the MMTV promoter in HeLa cells. Repression is DNA binding-independent as a deletion construct expressing the NH2-terminal 160 residues of NFI-C represses but does not bind DNA. Repression by NFI-C is cell type-dependent and occurs in HeLa and COS-1 cells but not 293 or JEG-3 cells. NFI-C does not repress progesterone induction of the MMTV promoter in HeLa cells, suggesting that progesterone induction of the promoter differs mechanistically from glucocorticoid induction. NFI-C-mediated repression is alleviated by overexpression of glucocorticoid receptor (GR), suggesting that NFI-C represses the MMTV promoter by preventing GR function. However, repression by NFI-C occurs with only a subset of glucocorticoid-responsive promoters, as the chimeric NFIGREbeta-gal promoter that is activated by GR is not repressed by NFI-C. Since the coactivator proteins p300/CBP, SRC-1A, and RAC3 had previously been shown to function at steroid hormone-responsive promoters, we asked whether they could influence NFI-C-mediated repression of MMTV expression. Expression of p300/CBP or SRC-1A alleviates repression by NFI-C, whereas RAC3 has no effect. This abrogation of NFI-C-mediated repression by p300/CBP and SRC-1A suggests that repression by NFI-C may occur by interference with coactivator function at the MMTV promoter.
Animals↗
The kinetics of oocyte activation and dynamics of polar body formation in Calomys musculinus and Calomys laucha (Rodentia-Sigmodontinae).
The objective of this study was to determine whether Calomys laucha and Calomys musculinus superovulated oocytes undergo parthenogenetic activation following activation stimuli. Cumulus-intact or denuded oocytes were treated with medium containing ethanol (7%), medium containing strontium chloride, or medium alone. They were then incubated for 6-8 h to allow for activation. A group of oocytes was fixed immediately after maturation to serve as a control. The nuclear status of the oocytes was examined after staining with Hoechst 33342, to determine the timing of pronuclear progression from metaphase II to anaphase II or telophase II or to the pronuclear stage. The proportion of oocytes that underwent activation was higher for oocytes treated with ethanol or strontium chloride than in those incubated in medium alone, for the two species studied (p < 0.001). There was little evidence of spontaneous activation occurring in oocytes during the treatments. Most of the activated oocytes contained a single haploid pronucleus, but it was possible to find immediate cleavage and two pronuclei. The different classes of activated oocytes were cultured for 5 days. The type of activating treatment had a marked effect on the ability of the resulting C. musculinus and C. laucha parthenogenetic embryos to develop to the preimplantation stages. Incubation with ethanol produced only 8-cell embryos while the embryos induced with strontium chloride reached the blastocyst stage. This is the first report of parthenogenesis in C. musculinus and C. laucha. The ability of strontium ions to induce matured secondary oocytes to initiate parthenogenesis and obtain further development of Calomys provides opportunities to use Calomys oocytes in vitro and, therefore, to study the genetics, cell biology and virology of development.
Animals↗
Superovulation in vesper mice, Calomys laucha--an important biomedical model for hantavirus and arenavirus (Rodentia-Sigmodontinae).
Sigmodontine rodents are poorly studied and have not received much attention as a reproductive model. Renewed interest in the South American rodents has been stimulated by their link to endemic diseases that are transmitted to man. Calomys laucha acts as a reservoir of two dangerous viruses: an arenavirus named 'Junin virus', the aetiological agent of Argentinian haemorrhagic fever, and the hantavirus, both of which constitute serious sanitary problems. The aim of this study was to establish suitable conditions to superovulate the vesper mouse, Calomys laucha. We examined the hormonal doses, the time interval between hormones, the time-course of ovulation, and the effect of female age on the response to exogenous hormone administration. Female mice were injected with 5-5, 8-8 or 12-15 IU of PMSG/hCG, 48 h apart, at different age intervals (from 30 to > 120 days old). The best superovulation rate was obtained with 8-8 IU PMSG/hCG. Ovulation started about 10 h post-hCG and was completed during the next 4-5 h, and was achieved irrespectively from the oestrus cycle stage. The number of oocytes was influenced by the age of the females. The youngest females had only a superovulatory response. Females older than 61 days showed both ovulatory and superovulatory responses, although 91-120-day-old females had a high ovulatory response. Most of the oocytes (96.5%) recovered were morphologically normal. The genus Calomys constitutes a reproductive model completely different from conventional laboratory rodents.
Aging↗
Nuclear factor I (NFI) isoforms differentially activate simple versus complex NFI-responsive promoters.
Promoter-specific differences in the function of transcription factors play a central role in the regulation of gene expression. We have measured the maximal transcriptional activation potentials of nuclear factor I (NFI) proteins encoded by each of the four identified NFI genes (NFI-A, -B, -C, and -X) by transient transfection in JEG-3 cells using two model NFI-dependent promoters: 1) a simple chimeric promoter containing a single NFI-binding site upstream of the adenovirus major late promoter (NFI-Ad), and 2) the more complex mouse mammary tumor virus long terminal repeat promoter. The relative activation potentials for the NFI isoforms differed between the two promoters, with NFI-X being the strongest activator of NFI-Ad and NFI-B being the strongest activator of the MMTV promoter. To determine if these promoter-specific differences in activation potential were due to the presence of glucocorticoid response elements (GREs), we added GREs upstream of the NFI-binding site in NFI-Ad. NFI-X remains the strongest activator of the GRE containing simple promoter, indicating that differences in relative activation potential are not due solely to the presence of GREs. Since NFI proteins bind to DNA as dimers, we assessed the activation potentials of NFI heterodimers. Here, we show that NFI heterodimers have intermediate activation potentials compared with homodimers, demonstrating one potential mechanism by which different NFI proteins can regulate gene expression.
Animals↗
Offspring sex-ratio and reproductive performance in heterogametic females of the South American field mouse Akodon azarae. Reproduction in heterogametic Akodon azarae females.
We have compared the breeding performance of homogametic (XX) and heterogametic (XY*) females of the South American sigmodontine rodent Akodon azarae under laboratory conditions. XY* females showed an enhanced reproductive performance when compared with normal, XX, females. The XY* females had a longer reproductive lifespan. They started to reproduce early, had more frequent litters, and stopped reproduction later than XX females. Their progeny showed a biased 1:2 male:female sex ratio which may be explained by the early loss of YY* zygotes after fertilization. However, litter size at birth was similar both in XY* and XX females, and no difference in ovulation rate was detected between them. This indicates that an "automatic" rather than an "evolved" reproductive compensation mechanism may be acting in heterogametic females. A separate study has shown that self-synapsis of both the X and Y* chromosomes takes place during meiosis, allowing the oocytes to escape from functional deterioration. It is suggested that self-synapsis and "automatic" reproductive compensation account for the preservation of fertility in heterogametic females in spite of the heteromorphic sex chromosomes and the early embryo loss they experience. However, these mechanisms do not account for the enhancement of reproductive lifespan. The possibility that an intrauterine position phenomenon is acting in A. azarae is discussed.
Animals↗
Repetitive calcium stimuli drive meiotic resumption and pronuclear development during mouse oocyte activation.
Freshly ovulated (12 hr post hCG) F1 (C57BL/6 x CBA) hybrid mouse oocytes were parthenogenetically activated by repetitive elevation of Ca2+ induced by carefully controlled electrical pulses. Different patterns of stimulation were employed to examine the role of repetitive calcium changes on meiotic resumption and pronuclear development. In the first series of experiments oocytes received 33 electrical pulses of 1.8 kV/cm delivered every 4 min. The pulse duration decreased according to a negative exponential equation from a 900-microseconds first pulse to give a total pulse duration of 18.721 msec. The strength of calcium stimuli was varied by changing the concentration of CaCl2 in the medium. Ninety-eight percent of the oocytes stimulated with 12 microM calcium extruded the second polar body by the end of treatment and 92% completed pronuclear formation between 3.5 and 8 hr after the first pulse. For higher or lower Ca2+ concentrations the proportion of oocytes developing pronuclei decreased; the timing of pronuclear formation was retarded and the majority of oocytes failed to form a pronucleus after extrusion of the second polar body. In the second series of experiments, the strength of the calcium stimuli was modulated by changing the duration of the 33 electrical pulses given in the presence of 12 microM calcium. By increasing the total pulse duration to 33.958 msec, 100% of the oocytes activated and completed pronuclear formation between 3 and 5 hr after the first electric pulse. Stimulation protocols of lower total pulse duration (less than 18.721 msec) gave rise to high rates of partial activation (up to 95%). Examination of these partially activated oocytes showed metaphases with haploid sets of chromatids characteristic of third meiotic metaphase arrest. The results indicate that repetitive calcium stimuli can regulate the rate and extent of meiotic resumption and the time course of pronuclear formation during mouse oocyte activation. They suggest that meiotic resumption in mammalian oocytes is regulated by the amplitude and frequency of cytosolic calcium oscillations induced by the activating stimulus.
Animals↗
The evolution of eutherian spermatozoa and underlying selective forces: female selection and sperm competition.
We have examined sperm morphology and dimensions in Eutherian mammals. In most Eutherians, sperm heads are round or oval and spermatozoa have short tails (average sperm length about 65 microns; range = 33-121 microns). Rodents, however, clearly depart from the typical Eutherian pattern in that they show a broad array of head morphs and an extreme range of sperm dimensions (35-250 microns). In order to trace the evolutionary changes that rodent sperm have undergone, we have used phylogenetic relationships based on biogeographical, morphological, chromosomal and genic data, and we have superimposed onto them the information available on sperm traits. Analyses were carried out for five rodent groups on which enough information was available. The evolutionary trends which emerged from these studies have two main points in common: throughout evolution spermatozoa have become enlarged and morphologically more complex, and this process seems to have taken place independently in different lineages. A general model was developed which outlines the different evolutionary pathways that rodent sperm have undergone. The adaptive significance of the increase in head complexity and the elongation of the sperm tail remains obscure. We have integrated information from evolutionary, physiological and behavioural studies to address this issue. We argue that two main selective forces may have favoured these changes: female selection within the reproductive tract and sperm competition. The female tract represents a formidable barrier for spermatozoa and its provides an environment where numerous interactions take place. The extent of these barriers and the complexity of these poorly understood interactions suggest that females may be exercising a strong selection, which may enable them to favour particular types of spermatozoa or ejaculates from particular males. Throughout their evolution males must have evolved adaptations to overcome these barriers, and the conflicting interests of choosy females. Sperm competition is a potent evolutionary force among mammals, which has influenced not only the evolution of sperm numbers but also changes in sperm dimensions. Thus, sperm competition has favoured the elongation of the sperm tail, which has led to the attainment of faster swimming speed, an important factor when sperm from rival males compete to reach the ova first.
Animals↗
Cyclic AMP inhibits fibronectin gene expression in a newly developed granulosa cell line by a mechanism that suppresses cAMP-responsive element-dependent transcriptional activation.
The main role of the ovarian granulosa cells is to nurse the oocyte and to produce estradiol and progesterone upon stimulation by gonadotropins. In fact, follicle-stimulating hormone (FSH) and luteinizing hormone control the expression of several genes during granulosa cell differentiation via cyclic AMP-dependent phosphorylations. Cyclic AMP stimulates transcription of genes that carry the cAMP-responsive element (CRE,5'TGACGTCA3') in their promoters. The fibronectin (FN) gene contains one CRE sequence at position -170. However, gonadotropins and cAMP inhibit FN gene expression in granulosa cells. To study the mechanism of the inhibition we developed a bovine granulosa cell line (BGC-1) that synthesizes estradiol in response to FSH and in which FSH and dibutyryl cAMP specifically decrease FN synthesis and its mRNA levels. The inhibitory effect (a) is not due to an alteration in FN mRNA stability, (b) requires upstream sequences other than CRE, located between positions -510 and -223, that are able to bind granulosa cell nuclear proteins, (c) is entirely dependent on the synthesis of intermediate proteins induced and or phosphorylated by cAMP, and (d) effectively suppresses the CRE-dependent transcriptional activation.
Animals↗
Vertical transmission of Junin virus in experimentally infected adult Calomys musculinus.
The response to infection with Junin virus, wild strain Cba An 9446, and the antenatal and postnatal transmission of the pathogen in its natural host, Calomys musculinus, were studied. Intranasal infection in adult animals (90-120 days) did not produce mortality or illness during the 150-day period of observation. From day 21 to 150 after infection, 50% of the animals showed viral persistence with shedding of virus in both urine and saliva. The remaining half became seropositive, and no infectious virus was recovered from them. Although the virus did not infect fetuses during gestation, 50% of weaned pups nursed by viremic mothers were infected. Neither persistence nor immunologic response altered the reproductive pattern of the animals. The absence of reproductive failure in the infected host and the efficiency of postnatal transfer of Junin virus indicate that vertical transmission could contribute to the viral maintenance over time.
Animals↗
Meiotic behavior of gonosomically variant females of Akodon azarae (Rodentia, Cricetidae).
The meiotic behavior of sex chromosomes has been investigated in variant females of Akodon azarae, both in pachytene oocytes and metaphase I. In somatic cells, these females have a heteromorphic sex pair, in which the minor chromosome has been previously interpreted as a major deletion of the long arm of the X chromosome (dX). After microspreading for synaptonemal complex analysis, pachytene oocytes show two axes of very different lengths (100:17.1), which correspond to the sex chromosomes X and dX. True synapsis is abnormally restricted (43.3%) between these sex chromosomes; on the other hand, self-synapsis of both the X and dX chromosomes is frequent (60%). Single, nonsynapsed axes or axial segments are thickened. Strong chromatin condensation occurs around nonsynapsed axes or axial segments, giving many of these sex pairs an appearance similar to an XY body ("sex vesicle"). The minor gonosome axis differs from that of the Y chromosome of male meiosis, as the former is shorter (relative to the X) and has a different synaptic behavior. In 17 metaphases I from XdX variant females, only heteromorphic, end-to-end joined sex pairs were observed. These variant females differ from the variant females of the wood lemming Myopus schisticolor in several respects, but a similar mechanism seems to be prevalent in other species of the genus Akodon. Self-synapsis of unequal gonosomes in oocytes is assumed as an escape from functional deterioration, following the hypothesis put forward by others.
Animals↗
Is vertical transmission sufficient to maintain Junin virus in nature?
The quantitative contribution of vertical transmission to the prevalence rate of Junin virus infection in subsequent generations of its natural reservoir, Calomys musculinus, was analysed. Data on mortality and reproduction of C. musculinus infected at birth with a wild strain of Junin virus were used to estimate the infection-dependent relative survival rate (beta = 0.4849) and relative fertility of the infected host (alpha = 0.2088). Prevalence rates of infection, obtained by mathematical simulation in optimal conditions of vertical transfer, dropped steadily to zero in a few generations. Vertical transmission was found to be insufficient to overcome the effect of highly depressed survival and fertility of the infected host and maintain a stabilized prevalence of Junin virus infection in successive generations; this suggested that viral maintenance is mainly dependent upon horizontal transmission.
Animals↗
Effect of persistent infection with Junin virus on growth and reproduction of its natural reservoir, Calomys musculinus.
The effect of infection with Junin virus on growth and reproduction of its natural reservoir, Calomys musculinus, was studied. Eighty-five C. musculinus were inoculated intranasally at birth with 100 TCID50 of Cba An 9446 strain of Junin virus and observed for 480 days. No clinical signs of neurologic illness were registered. Infected animals showed an increased mortality rate of up to 70% between days 24-40 post-infection. This period of high mortality was preceded by low weight gain during lactation and registered until 60 days. From day 14 post-infection until day 480, Junin virus was recovered from blood, urine, and oral swab in all animals checked at any time. By day 480 post-infection, 100% of survivors showed widespread viral dissemination in brain, spleen, kidneys, and salivary glands. There was marked reduction in reproductive efficiency among infected animals. Out of 15 mating pairs, 2 (13.3%) littered at least once compared to 60% in the control group. The reduction of fertility and the altered survival rate of Junin virus-infected C. musculinus indicate that vertical transmission mechanisms per se are insufficient to maintain the infection in successive generations in the absence of horizontal transmission.
Animals↗
Cross fertilization in vivo and in vitro between three species of vesper mice, Calomys (Rodentia, Cricetidae).
Cross fertilization was tested between oocytes of Calomys callidus and spermatozoa from C. callidus, C. musculinus and C. laucha by both in vivo and in vitro insemination. After in vivo and in vitro insemination, respectively, percentages of oocytes fertilized were 68.8 and 46.6 (C. callidus X C. callidus), 20.3 and 12.7 (C. callidus X C. musculinus), 26.7 and 4.3 (C. callidus X C. laucha). Thus, the percentages obtained after in vitro insemination were always lower than those obtained with in vivo insemination. It was found that 23.9% and 44.4% of two-cell hybrid embryos were present in oviducts 30 hr after in vivo insemination of C. callidus females with C. musculinus or C. laucha spermatozoa, respectively. At a later stage (56 hr postinsemination), development did not progress further, and abnormal embryos were found both at 30 and 56 hr postinsemination, suggesting some kind of cleavage arrest or degeneration of the embryos. We suggest that fertilization is not strictly species-specific, at least among the species we studied, but that there are some factors that reduce the efficiency of interspecific fertilization.
Animals↗