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S Garagna

Publications and source records attributed to S Garagna.

At least 37 records · Page 2Linked to original sources
PubMed1998

Timing of gene expression and oolemma localization of mouse alpha6 and beta1 integrin subunits during oogenesis.

The sperm antigen fertilin alpha/beta and the integrin complex alpha6beta1 present on the oolemma are two of the most promising candidates to mediate gamete interaction. During growth, the plasma membrane of both hamster and mouse zona-free oocytes acquires the capacity to fuse with acrosome-reacted sperm when oocytes reach the size of 25-30 microm in diameter, suggesting changes in the membrane molecular composition. The present study has two aims: to determine the timing of (1) gene expression of alpha6 and beta1 integrins and (2) localization of these integrin subunits on the plasma membrane in primordial germ cells and in oocytes during oogenesis. We found that both alpha6 and beta1 genes are expressed in female germ cells during all the stages of development analyzed, from 10.5 to 18.5 d.p. c., during oocyte growth, and in ovulated eggs. The alternatively spliced isoform alpha6B is expressed from 10.5 d.p.c., whereas alpha6A begins to be expressed at 12.5 d.p.c., suggesting a different role for the two variants. In situ immunodetection of alpha6 or beta1 shows a ring of fluorescence on the female germ cell plasma membrane for both integrins at 10.5 d.p.c., then the fluorescent signal becomes undetectable at 12.5 d.p.c. to reappear again, this time with a patchy distribution, at 18.5 d.p.c. This pattern of localization is maintained in oocytes isolated from newborn individuals and only when oocytes during growth reach the size of about 25-30 microm in diameter does the fluorescence become homogenous all around the whole oocyte surface. These data, although not conclusive, support the hypothesis of an involvement of alpha6 and beta1 integrins in sperm-egg fusion.

Animals↗
PubMed1998

Analysis of aneuploidy rate in antral and ovulated mouse oocytes during female aging.

Two forms of oocytes termed SN (surrounded nucleolus) and NSN (nonsurrounded nucleolus) differing for the spatial distribution of nuclear and nucleolar-associated chromatin have been described within the antral compartment of the ovary of a number of mammals. The biological significance of these two kind of oocytes is as yet not completely clear. In previous studies we have shown that prior to ovulation, mouse SN oocytes isolated from the antral compartment, matured and fertilized in vitro have a far better meiotic and developmental competence than NSN oocytes. Immediately after ovulation SN and NSN oocytes remaining in the antral compartment do not develop beyond the 2-cell stage. To further examine the correlation between chromatin distribution and meiotic competence of mouse antral oocytes, in the present study we have analyzed chromosome segregation at the first meiotic division in antral (SN and NSN) and in ovulated oocytes. SN and NSN oocytes were isolated before (48 h post PMSG injection) or after (15 h post-hCG injection) ovulation from ovaries of females of increasing age, they were cultured in vitro to metaphase II, and their aneuploidy rate was examined. Comparison of data obtained before and after ovulation highlights two main points: 1. Following ovulation a statistically significant increase of aneuploidy is observed in antral oocytes in most age groups and it is attributable to SN oocytes. 2. The aneuploidy rate of ovulated oocytes does not increase during female aging. We have found a correlation between chromatin distribution, hormonal status, and the incidence of aneuploidy during the oocyte first meiotic division.

Aging↗
PubMed1998

Meiotic and developmental competence of mouse antral oocytes.

Mouse antral oocytes show two different patterns of chromatin organization, defining oocytes with or without chromatin surrounding the nucleolus (SN: surrounded nucleus; NSN: not surrounded nucleus). We have previously shown that upon injection of eCG, NSN antral oocytes shift towards the SN kind of chromatin organization. We hypothesized that these newly formed SN oocytes were those that would have been ovulated after an ovulatory stimulus. The main objective of this study was to investigate the meiotic and developmental competence of these two types of oocytes. SN and NSN antral oocytes were isolated after i.p. administration of eCG + hCG or eCG-only, in vitro-cultured until completion of metaphase II, and inseminated with capacitated spermatozoa; and their development to the 4-cell stage was examined. This study demonstrates 1) that SN and NSN oocytes isolated after injection of eCG + hCG are capable of embryonic development, but not beyond the 2-cell stage; and 2) that SN and NSN oocytes isolated after injection of eCG-only are capable of developing to the 2-cell stage, but a significantly higher number (11.9%) of SN oocytes than NSN oocytes (1.5%) reach the 4-cell stage. SN- and NSN-like oocytes have also been described in the antral compartment of human, rat, monkey, pig, and bovine ovaries. The findings reported in this paper may contribute to improved procedures for in vitro fertilization of humans and farm animals.

Animals↗
PubMed1998

Six-year-old archival chromosome preparations are still good biological reagents for repeated primed in situ labelling (rPRINS).

Six-year-old chromosome preparations of Eulemur coronatus were used for in situ localization of highly repetitive DNA sequences. The application of the repeated primed in situ labelling (rPRINS) technique allowed the detection of positive signals whereas conventional fluorescence in situ hybridization gave negative results on the same archival preparations. This paper reveals the possibility of rescuing archival chromosome preparations for both clinical and comparative cytogenetics. Moreover, the chromosomal localization of the ECO-SAT 503 bp highly repetitive DNA sequences were found to localize in the pericentromeric region of every chromosome, with the exception of chromosome 9.

Animals↗
PubMed1997

Non-telomeric chromosome localization of (TTAGGG)n repeats in the genus Eulemur.

The chromosomal distribution of the (TTAGGG)n telomeric repetitive sequences was studied in the Malagasy species Eulemur fulvus fulvus (2n = 60), Eulemur rubriventer (2n = 50), Eulemur coronatus (2n = 46) and Eulemur macaco (2n = 44). These sequences hybridize to the telomeres of all chromosomes of the four species and also to the pericentromeres of all chromosomes of E. fulvus, E. coronatus and E. macaco, with the exception of the pericentromeres of E. coronatus and E. macaco chromosomes 9, the homeologous E. fulvus chromosomes 2 and E. macaco chromosomes 1. In E. rubriventer only a very weak signal was detected at the pericentromeres of a few chromosomes. In E. fulvus, E. coronatus and E. macaco, non-telomeric (TTAGGG)n sequences collocalize with constitutive heterochromatin. The interspecific differences of the hybridization pattern of (TTAGGG)n sequences at the pericentromeres suggest that E. rubriventer branched off the common trunk before amplification of endogenous (TTAGGG)n sequences occurred in pericentromeric regions.

Animals↗
PubMed1997

Genome composition in Venezuelan spiny-rats of the genus Proechimys(Rodentia, Echimyidae). I. Genome size, C-heterochromatin and repetitive DNAs in situ hybridization patterns.

The genome sizes of the Venezuelan spiny-rats Proechimys guairae guairae (2n = 48) and P. trinitatis (2n = 62) were evaluated and proved to be 12.5 +/- 0.5 pg and 12.6 +/- 0.3 pg respectively, the highest so far recorded among mammals; also the C-heterochromatin (32.7%, Coefficient of Variation [CV] 3.8 and 35.8%, CV 4.4) and GC (44.2%, CV 2.7 and 43.6%, CV 2.9) contents are very high. Highly repetitive (rep) DNAs were isolated from restriction enzyme digested genomic DNAs of both species. The intra- and inter-specific chromosomal allocations of these rep DNAs were analyzed by direct and cross-hybridizations. Results show that the two genomes harbour several rep DNA families which show both species-specificity and interspecific relatedness in their in situ hybridization patterns. The rep DNA families show an equilocal distribution at both the pericentromeric areas of all chromosomes and in the whole arms of two pairs of the uniarmed group, suggesting co-evolution of the rep DNAs. P. g. guairae BamHI digested DNA, when cloned and sequenced, proved to consist of a long "composite" unit (1,239 bp) containing two copies of each of 75-bp and 110-bp internal subrepeats. Karyotype restructuring between P. g. guairae and P. trinitatis, mainly due to Robertsonian changes, was accompanied by slight intra- and intergenomic movements of the putative satellite DNA families within stable genome sizes and C-heterochromatin contents. We discuss the findings obtained in Proechimys in the light of those regarding the kangaroo rat, the pocket gopher and the house mouse; they support the idea that karyotype restructuring could be the expression of molecular driven events of rep DNA amplification and homogenisation through non-homologous chromosomes.

Animals↗
PubMed1995

Chromatin organization during mouse oocyte growth.

We investigated the changes in the organization of oocyte nuclear chromatin and nucleolar-associated chromatin throughout folliculogenesis. Zona-free oocytes were isolated from ovaries, grouped into seven classes according to size and chromatin organization, and analyzed after staining with Hoechst 33342. We show that oocyte differentiation from the dictyate stage to the conclusion of maturation is associated with either of two chromatin configurations. Initially, all oocytes are in the NSN configuration (nonsurrounded nucleolus oocytes; characterized by a Hoechst positive-chromatin pattern of small clumps forming a network on the nuclear surface, with a nucleolus nonsurrounded by chromatin). While growing some of these NSN oocytes continue their development in the NSN configuration, whereas others shift (from class IV on) into the SN configuration (surrounded nucleolus oocytes; characterized by a threadlike chromatin organization that may partially surround the nucleolus or project towards the nuclear periphery). The percentage of SN oocytes increases both with increasing size of the oocyte (class I-III, 10-40 microns in diameter: 100% NSN vs. 0% SN; class VII 70-80 microns in diameter: 47.3% NSN vs. 52.3 SN, in 4-6-week-old females), and with aging (class VII: 94.1% NSN vs. 5.9% SN in 2-week-old females; 11.8% NSN vs. 8.2% SN in 56-week-old females). Further, we suggest as a working hypothesis that those oocytes that switch to the SN chromatin organization early in maturation may not be ovulated, even though this particular chromatin structure normally occurs just prior to ovulation.

Animals↗
PubMed1995

Robertsonian metacentrics of the house mouse lose telomeric sequences but retain some minor satellite DNA in the pericentromeric area.

A combination of cytogenetic and molecular biology techniques were used to study the molecular composition and organisation of the pericentromeric regions of house mouse metacentric chromosomes, the products of Robertsonian (Rb) translocations between telocentrics. Regardless of whether mitotic or meiotic preparations were used, in situ hybridisation failed to reveal pericentromeric telomeric sequences on any of the Rb chromosomes, while all metacentrics retained detectable, although reduced (average 50 kb), amounts of minor satellite DNA in the vicinity of their centromeres. These results were supported by slot blot hybridisation which indicated that mice with 2n=22 Rb chromosomes have 65% of telomeric sequences (which are allocated to the distal telomeres of both Rb and telocentric chromosomes and to the proximal telomeres of telocentrics) and 15% the amount of minor satellite, compared with mice with 2n=40 all-telocentric chromosomes. Pulsed field gel electrophoresis and Southern analysis of DNA from Rb mice showed that the size of the telomeric arrays is similar to that of mice with all-telocentric chromosomes and that the minor satellite sequences were hybridising to larger fragments incorporating major satellite DNA. Since the telomeric sequences are closer to the physical end of the chromosome than the minor satellite sequences, the absence of telomeric sequences and the reduced amount of minor satellite sequences at the pericentromeric region of the Rb metacentrics suggest that the breakpoints for the Rb translocation occur very close to the minor satellite-major satellite border. Moreover, it is likely that the minor satellite is required for centromeric function, 50-67 kb being enough DNA to organise one centromere with a functionally active kinetochore.

Animals↗
PubMed1995

Study of genome organization by DNA cytochemistry and fluorescence imaging: a review.

DNA analysis by quantitative cytochemistry has been a widely exploited approach to investigate chromatin superstructural changes in relation to cell function and cell cycle progress. To this aim, a number of dyes (especially fluorochromes) for nucleic acids have been used, exhibiting different peculiarities, in terms of both binding mechanism and base specificity. Less attention has been paid to the application of different DNA staining techniques for studying possible differences in genome organization among different taxa. The purpose of this paper is to review and discuss the present reports in the literature, concerning the cytochemical analysis of genome organization, in a comparative perspective. Special attention is given to the integration of quantitative studies based on cytofluorometric and imaging techniques.

Animals↗
PubMed1993

Genome distribution, chromosomal allocation, and organization of the major and minor satellite DNAs in 11 species and subspecies of the genus Mus.

We compared the genome distribution, chromosomal allocation, and organization of the major and minor satellite DNAs (satDNAs) in 11 species and subspecies of the genus Mus. Southern blot analysis of the major and minor satDNAs showed similar fragment profiles in all 11 species, with the exception of M. cervicolor and M. cookii for the major satDNAs and M. caroli, M. cervicolor, and M. cookii for the minor satDNAs, where these sequences could not be detected by the probes used. In situ hybridization of the major and minor satDNA probes revealed chromosome-specific allocations of these sequences with quantitative species-specific patterns. Fluorometric analysis of the organization of the satellite sequences suggested that in the M. domesticus genome satDNA sequences are clustered in tandem repeats that are longer than those present in other Mus genomes. When compared with the other Mus genomes so far studied, the domesticus genome shows the highest quantity of satDNA sequences with a long-range organization of satDNA sequences.

Animals↗
PubMed1993

Further examination of the kinetics of gonadal development in XY female mice.

Y-chromosomes derived from house mouse populations of Mus domesticus are able to lead to the formation of XY females (i.e. hermaphrodite individuals with both ovarian and testicular tissues) when inserted, by appropriate crosses, into the C57BL/6J genome, which usually carries the Mus musculus Y chromosome. An abnormal interaction between the testis-determining gene on the Y (of domesticus origin) and an autosomal recessive allele (of musculus origin) needed for testicular formation is the likely explanation of this phenomenon. In the present paper we analyse the histological kinetics of gonadal development in hermaphrodites obtained by introducing the Y chromosomes from wild living house mice [from Zagreb, (Croatia) and Hohenentringen (Southern Germany)] into the C57BL/6J genome. Among 88 individuals, 9 XY hermaphrodites were detected, 6 before birth and 3 afterwards. The percentage of the ovarian-type tissues in the ovotestes fell drastically between day 18 of fetal life and day 20 after birth. These findings corroborate and extend those already published for other Y domesticus chromosomes and stress the possible role of Y chromosome differentiation in speciation processes.

Aging↗
PubMed1990

Protamine amount and cross linking in mouse teratospermatozoa and aneuploid spermatozoa.

The level of SH-group oxidation in spermatozoa from the cauda epididymis was measured by a cytofluorometric method in chromosomally normal mice and two chromosome mutants. The first one, a tertiary trisomic karyotype (Ts(1(13]7OH), is characterized by severe oligospermia and high levels (approximately 75%) of malformed spermatozoa. The second, a hybrid between two European feral mouse stocks, is heterozygous for multiple Robertsonian translocations and produces exclusively aneuploid spermatozoa. Neither the severe teratospermiogenesis nor the severe aneuploidy was reflected in total SH-group fluorescence values nor in free SH-group fluorescence. It is concluded that both the production of protamines and protamine cross linking by S-S bridge formation are rather autonomous processes during spermatogenesis because 1) the increased DNA variance of the aneuploid spermatozoa is not reflected in an increased variance of the total and free SH-groups, 2) aneuploidy for the protamine gene carrying chromosome 16 is not reflected by the SH-group values for individual spermatozoa, and 3) protamine production and cross linking are independent of the mild to severe terataspermiogenesis in the tertiary trisomic karyotype.

Aneuploidy↗
PubMed1990

Differences in the organization and chromosomal allocation of satellite DNA between the European long tailed house mice Mus domesticus and Mus musculus.

We compared the organization of satellite DNA (stDNA) and its chromosomal allocation in Mus domesticus and in Mus musculus. The two stDNAs show similar restriction fragment profiles after digestion (probed with M. domesticus stDNA) with some endonucleases of which restriction sequences are present in the 230-240 bp repetitive unit of the M. domesticus stDNA. In contrast, EcoRI digestion reveals that M. musculus stDNA lacks most of the GAATTC restriction sites, particularly at the level of the half-monomer. The chromosome distribution of stDNA (revealed by an M. domesticus stDNA probe) shows different patterns in the M. domesticus and M. musculus karyotypes, with about 60% of M. domesticus stDNA retained in the M. musculus genome. It is particularly noteworthy that the pericentromeric regions of M. musculus chromosomes 1 and X are totally devoid of M. domesticus stDNA sequences. In both groups, the differences in energy transfer between the stDNA-bound fluorochromes Hoechst 33258 and propidium iodide suggest that AT-rich repeated sequences have a much more clustered array in the M. domesticus stDNA, as if they are organized in tandem repeats longer than those of M. musculus. Considering the data as a whole, it seems likely that the evolutionary paths of the two stDNAs diverged after the generation of the ancestral 230-240 bp stDNA repetitive unit through the amplification, in the M. domesticus genome, of a family repeat which included the EcoRI GAATTC restriction sequence.

Animals↗
PubMed1990

Kinetics of oogenesis in mice heterozygous for Robertsonian translocation.

The total number of oocytes at different postmating time intervals (18-40 days) was determined in mice homozygous and heterozygous for different Robertsonian (Rb) translocations, of both laboratory and feral origin. The number of oocytes was lower in heterozygous than in homozygous mice throughout the period studied. Independently of the genetic background (i.e. laboratory or feral), structural heterozygosity had a progressive detrimental effect on oocyte numbers: open, or chain diakinetic configurations had a greater detrimental effect than close, or ring, configurations. The genetic background, however, affected the ovarian constitution in terms of the total number of germ cells, which are more numerous in laboratory than in feral mice. The kinetics of oogenesis seems to be faster in feral than in laboratory mice. At the light of the data here presented, and of those already available from the literature on male and female gametogenesis in conditions of structural heterozygosity, it appears that factors other than unsaturation of pairing sites or interference with pachytene X-chromosome inactivation have to be considered. In the wild, the reduced oocyte numbers in Rb heterozygous female can contribute to the retention of isolated populations in contact zones.

Animals↗
PubMed1989

Spermatogenesis in heterozygotes for Robertsonian chromosomal rearrangements from natural populations of the common shrew, Sorex araneus.

Twenty-two adult male common shrews were collected from 5 sites in the vicinity of Oxford (UK) close to the zone of hybridization between two karyotypic races. The shrews were subdivided into 3 karyotypic categories: homozygotes, simple Robertsonian heterozygotes (which form one or more trivalents at prophase I of meiosis) and complex Robertsonian heterozygotes (which form a quadrivalent). The ratio of primary spermatocytes to round spermatids was determined from transverse sections of seminiferous tubules, to provide an indication of germ cell death. In no individual was there severe germ cells loss. Homozygotes had the highest mean spermatocyte: spermatid ratio and complex heterozygotes the lowest, but there was substantial individual variation and the differences were not significant. Complex heterozygotes also had a higher proportion of defective seminiferous tubules and lower testis weights than did other categories and it is reasonable to propose that, as a population, complex heterozygotes had reduced fitness relative to other categories on the basis of spermatogenic performance. However, there is no evidence from studies of spermatogenesis that simple Robertsonian heterozygotes are less fit than homozygotes.

Animals↗
PubMed1988

Chromatin topology during the transformation of the mouse sperm nucleus into pronucleus in vivo.

Time relationships of sperm chromatin dispersion and sperm nucleoprotein replacement have been studied in vivo, by an in situ cytochemical approach. We used the Feulgen reaction to reveal DNA, which allow us to record both processes simultaneously, on the basis of the return after fertilization to haploid Feulgen values after sperm nucleoprotein replacement with somatic histones. We have shown that sperm nucleoprotein replacement occurs at around anaphase II, whereas sperm chromatin dispersion is massive between the anaphase and telophase II oocyte phases. The morphological pattern of sperm chromatin dispersion supports the idea that the process involves the whole sperm chromatin mass simultaneously, with the region located between the implantation fossa and the postacrosomial region the last to swell.

Anaphase↗