19 research outputs found
Manipulation of gene expression in early mouse embryo
Maternal (oocyte) and embryonic programmes of hypoxanthine phosphoribosyl transferase (HPRT) and adenine phosphoribosyl transferase (APRT) gene expression have been investigated in mouse oocytes and during preimplantation development. The onset of the embryonic HPRT gene occurs following fertilization, or parthenogenetic activation, before the 4-cell stage. The oocyte-determined increase in HPRT activity due to preformed mRNA continues in aging oocytes, as well as in fertilized or activated eggs, i.e. irrespective of the initiation of embryonic development. Attempts were made to determine whether there is a detectable time difference in the onset of maternal and paternal genomes by assaying the onset of embryo- coded HPRT activity in embryos of different maternal and paternal X-chromosome constitution, but due to difficulties in comparable staging of embryos, no definite conclusion could be drawn. The expression of an exogenously introduced HPRT minigene has been monitored throughout preimplantation development. The embryos injected with supercoiled HPRT minigene showed an approximately twofold increase in HPRT activity at the 2-cell stage compared with control uninjected embryos. Linear minigene DNA was less efficient in giving active enzyme. The efficacy of three different promoters were studied in 2-cell mouse embryos using the expression of the HPRT minigene as a reporter function. The mouse HPRT promoter and the uninduced mouse metallothionein-1 (MT-1) promoter functioned equally well whereas the viral SV40 promoter did not allow HPRT expression. The mouse MT-1 promoter linked to the HPRT minigene allowed induction of HPRT gene expression in mouse embryos cultured in the presence of cadmium. The inhibition of enzyme expression from injected minigene DNA is mediated by simultaneous injection of a fivefold molar excess of HPRT antisense DNA. The same negation of exogenous HPRT activity was observed with simultaneous injection of HPRT exon-1 antisense DNA. The use of an inducible HPRT antisense construct achieved repression of gene activity with equivalent molar amounts of antisense to the sense molecules. Transgenic mice were produced with an antisense HPRT minigene construct attached to the inducible mouse MT-l promoter. The prospect of "cancelling" the endogenous "sense" gene activity in these mice at a specific stage of development by applying the induction stimulus is discussed
Vasoactive Intestinal Peptide and Its Receptors in Human Ovarian Cortical Follicles
BACKGROUND: Ovarian cryopreservation is one option for fertility preservation in patients with cancer. The danger of reseeding malignancies could be eliminated by in vitro maturation of primordial follicles from the frozen-thawed tissue. However, the development of this system is hindered by uncertainties regarding factors that activate primordial follicles. Neuronal growth factors such as vasoactive intestinal peptide (VIP) play important roles in early mammalian folliculogenesis. There are no data on the expression of VIP and its vasoactive intestinal peptide pituitary adenylate cyclase 1 and 2 receptors (VPAC1-R and VPAC2-R) in human preantral follicles. METHODOLOGY/PRINCIPAL FINDINGS: Tissue samples from 14 human fetal ovaries and 40 ovaries from girls/women were prepared to test for the expression of VIP, VPAC1-R, and VPAC2-R on the protein (immunohistochemisty) and mRNA (reverse transcription polymerase chain reaction) levels. Immunohistochemistry staining was mostly weak, especially in fetal samples. The VIP protein was identified in oocytes and granulosa cells (GCs) in the fetal samples from 22 gestational weeks (GW) onwards. In girls/women, VIP follicular staining (oocytes and GCs) was identified in 45% of samples. VPAC1-R protein was identified in follicles in all fetal samples from 22GW onwards and in 63% of the samples from girls/women (GC staining only in 40%). VPAC2-R protein was identified in follicles in 33% of fetal samples and 47% of the samples from girls/women. The mRNA transcripts for VIP, VPAC1-R, and VPAC2-R were identified in ovarian extracts from fetuses and women. CONCLUSIONS: VIP and its two receptors are expressed in human ovarian preantral follicles. However, their weak staining suggests they have limited roles in early follicular growth. To elucidate if VIP activates human primordial follicles, it should be added to the culture medium
The Role of Mitochondrial DNA Copy Number in Mammalian Fertility1
Mammalian mitochondrial DNA (mtDNA) is a small, maternally inherited genome that codes for 13 essential proteins in the respiratory chain. Mature oocytes contain more than 150â000 copies of mtDNA, at least an order of magnitude greater than the number in most somatic cells, but sperm contain only approximately 100 copies. Mitochondrial oxidative phosphorylation has been suggested to be an important determinant of oocyte quality and sperm motility; however, the functional significance of the high mtDNA copy number in oocytes, and of the low copy number in sperm, remains unclear. To investigate the effects of mtDNA copy number on fertility, we genetically manipulated mtDNA copy number in the mouse by deleting one copy of Tfam, an essential component of the mitochondrial nucleoid, at different stages of germline development. We show that males can tolerate at least a threefold reduction in mtDNA copy number in their sperm without impaired fertility, and in fact, they preferentially transmit a deleted Tfam allele. Surprisingly, oocytes with as few as 4000 copies of mtDNA can be fertilized and progress normally through preimplantation development to the blastocyst stage. The mature oocyte, however, has a critical postimplantation developmental threshold of 40â000â50â000 copies of mtDNA in the mature oocyte. These observations suggest that the high mtDNA copy number in the mature oocyte is a genetic device designed to distribute mitochondria and mtDNAs to the cells of the early postimplantation embryo before mitochondrial biogenesis and mtDNA replication resumes, whereas down-regulation of mtDNA copy number is important for normal sperm function
Recurrent Triploid and Dispermic Conceptions in Patients with NLRP7 Mutations
To understand the mechanisms leading to hydatidiform mole formation in patients with NLRP7 mutations, we used a combination of various approaches to characterize five products of conception, from two patients, shown by flow cytometry to contain non-diploid cells. We demonstrate that four of these conceptions are triploid and two of them originated from fertilization with more than one sperm. We show that three of these triploid conceptions fulfill the histopathological criteria of partial hydatidiform mole and one fulfills the histopathological criteria of spontaneous abortion. Our data demonstrate that oocytes from one patient with NLRP7 mutations are not able to prevent polyspermic fertilization and highlight the importance of using several approaches to characterize the genetic complexity of molar tissues and reproductive wastage. Altogether, our previous and current data show the association of NLRP7 mutations with several types of hydatidiform moles and with triploid spontaneous abortions
Chromosome Segregation Analysis in Human Embryos Obtained from Couples Involving Male Carriers of Reciprocal or Robertsonian Translocation
<div><p>The objective of this study was to investigate the frequency and type of chromosome segregation patterns in cleavage stage embryos obtained from male carriers of Robertsonian (ROB) and reciprocal (REC) translocations undergoing preimplantation genetic diagnosis (PGD) at our reproductive center. We used FISH to analyze chromosome segregation in 308 day 3 cleavage stage embryos obtained from 26 patients. The percentage of embryos consistent with normal or balanced segregation (55.1% vs. 27.1%) and clinical pregnancy (62.5% vs. 19.2%) rates were higher in ROB than the REC translocation carriers. Involvement of non-acrocentric chromosome(s) or terminal breakpoint(s) in reciprocal translocations was associated with an increase in the percent of embryos consistent with adjacent 1 but with a decrease in 3â¶1 segregation. Similar results were obtained in the analysis of nontransferred embryos donated for research. 3â¶1 segregation was the most frequent segregation type in both day 3 (31%) and spare (35%) embryos obtained from carriers of t(11;22)(q23;q11), the only non-random REC with the same breakpoint reported in a large number of unrelated families mainly identified by the birth of a child with derivative chromosome 22. These results suggest that chromosome segregation patterns in day 3 and nontransferred embryos obtained from male translocation carriers vary with the type of translocation and involvement of acrocentric chromosome(s) or terminal breakpoint(s). These results should be helpful in estimating reproductive success in translocation carriers undergoing PGD.</p> </div
Percent segregation and number of embryos (in parenthesis) obtained from reciprocal translocation carriers classified based on presence of chromosomes with terminal breakpoints or acrocenteric chromosomes<sup>a</sup>.
a<p>Abbreviations used: ACRâ=âacrocentric chromosome(s) involved; NonACRâ=âno acrocentric chromosome(s) involved; TERâ=âchromosome(s) with terminal breakpoint(s) involved; NonTERâ=âno chromosome(s) with terminal breakpoint(s) involved; N/Bâ=âembryos consistent with normal or balanced chromosome complement; NSâ=âembryos without any known segregation type; N/Aâ=ânot applicable.</p>b<p>Total number of embryos.</p
Karyotype and reproductive history of the male translocation carriers.
a<p>Age of patient at the time of embryo biopsy averaged over the number of PGD cycles the couple undergone.</p>b<p>OSâ=âoligospermia, ASTâ=âasthenospermia, TRTâ=âteratozoospermia, TOPâ=âtermination of pregnancy.</p>c<p>This couple used donor eggs from 32 and 27 year old women in the two PGD cycles.</p
Chromosome segregation in day 3 cleavage stage and spare embryos obtained from male translocation carriers<sup>a</sup>.
<p><sup>a</sup>RECâ=âreciprocal translocation; ROBâ=âRobersonian translocation, N/Bâ=âchromosomally normal or balanced for the translocated chromosomes, Adj1â=âadjacent 1, Adj2â=âadjacent 2, NSâ=âno known segregation pattern detected, Polyâ=âpolyploid. An asterisk (*) denotes statistically significant differences (<i>P</i><0.05). The column labeled with â3â¶1â represents frequency of 3â¶0 segregants in ROB and 3â¶1 in REC. Normal/balanced spare embryos in ROB and REC carriers were not compared.</p
Clinical details of patients participated in this study categorized by the type of translocation<sup>a</sup>.
a<p>Abbreviations used: RECâ=âreciprocal translocation, ROBâ=âRobertsonian translocation, an asterisk (*) indicates statistical difference at <i>P</i><0.05 level.</p