Characterization of the full fragile X syndrome mutation in fetal gametes.

Fragile X syndrome results from the expansion of the CGG repeat in the FMR1 gene. Expansion has been suggested to be a postzygotic event with the germline protected. From an analysis of intact ovaries of full mutation fetuses, we now show that only full expansion alleles can be detected in oocytes (but in the unmethylated state). Similarly, the testes of a 13-week full mutation fetus show no evidence of premutations while a 17-week full mutation fetus exhibits some germ cells with attributes of premutations. These data discount the hypothesis that the germline is protected from full expansion and suggest full mutation contraction in the immature testis. Thus, full expansion may already exist in the maternal oocyte, or postzygotic expansion, if it occurs, arises quite early in development prior to germline segregation.

Partial zona dissection of the human oocyte: a nontraumatic method using micromanipulation to assist zona pellucida penetration.

Partial zona dissection (PZD), a method using mechanical force to open the human zona pellucida, and zona drilling, which uses acidic Tyrode's (AT) medium, were compared in 1-day-old oocytes prior to reinsemination. The incidences of monospermy and polyspermy were 13/54 (24%) and 14/54 (26%) following PZD and 6/46 (13%) and 8/46 (17%) following the use of AT medium. This compared favorably with conventional reinsemination: 15/161 (9%) monospermy and 4/161 (3%) polyspermy. Three of the 27 PZD embryos became blastocysts, while none of the AT-exposed embryos developed satisfactorily. Eleven male-factor couples had some of their oocytes randomly treated with PZD prior to insemination; each of the patients had non-micromanipulated control oocytes. Monospermic fertilization and cleavage (23/34; 68%) doubled (P less than 0.05) when PZD was compared with the control oocytes (10/30; 33%). Replacing two PZD and a single control embryo in two patients resulted in twin pregnancies. A third twin pregnancy was established following replacement of only micromanipulated embryos.

Embryonic development after microsurgical repair of polyspermic human zygotes.

Correction of polyspermy through pronucleus extraction in the absence of membrane relaxants was applied to 25 polyspermic human zygotes. Nine zygotes survived the procedure, and seven cleaved normally (two of which were fixed for chromosome analysis); two proceeded to compact and one cavitated. Eighteen of the polyspermic zygotes (14 with three pronuclei and 4 with four pronuclei) were obtained from zona pellucida-intact oocytes, and seven (1 five-pronucleate 3 four-pronucleate, and 3 three-pronucleate) from previously zona-drilled oocytes. Survival and cleavage occurred in all groups except in four- and five-pronucleate zona-drilled zygotes. Criteria used to identify male pronuclei were (1) pronucleus-associated sperm tails, (2) increased pronucleus size, and (3) greater distance (relative to female pronuclei) from the second polar body. Sperm tails were never seen and pronucleus size usually was identical. Therefore, the third criterion was used, although its reliability should be further evaluated. Until complete pronucleus removal techniques and reliable pronucleus selection criteria are perfected, embryo replacement after polyspermy correction could result in aneuploidy and molar pregnancy.

Partial zona dissection or subzonal sperm insertion: microsurgical fertilization alternatives based on evaluation of sperm and embryo morphology.

OBJECTIVE: To establish guidelines for application of partial zona dissection, subzonal sperm insertion, and regular in vitro fertilization (IVF) in severe male factor patients. DESIGN: Two studies were performed: partial zona dissection and IVF was applied in 57 couples during the first period, and subzonal sperm insertion was also applied in a second group of 47 couples. SETTING: Procedures were performed in an academic research environment. PATIENTS, PARTICIPANTS: Couples who failed fertilization previously, others not acceptable for IVF, and a third group in whom IVF was expected to fail. INTERVENTIONS: Oocytes were micromanipulated with either partial zona dissection or subzonal sperm insertion, or the zona pellucida was left intact. Embryos were replaced in patients prophylactically treated with methylprednisolone and antibiotics. MAIN OUTCOME MEASURES: Because several microsurgical fertilization techniques are now available, this study was performed to compare sperm parameters, embryo morphology, fertilization, and implantation rates after application of two successful micromanipulation procedures. RESULTS: Twenty-one pregnancies were established in 104 patients, 5 definitely from subzonal sperm insertion and 4 from partial zona dissection. Patients who failed IVF before had a similar chance of pregnancy after the use of micromanipulation, as first time patients (9/53 versus 12/51). In a subgroup of 15 patients who failed IVF with insufficient numbers of motile sperm, fertilization was significantly higher after subzonal sperm insertion. Partially zona-dissected embryos from couples with severe teratozoospermia (less than or equal to 5% normal forms; strict criteria) had significantly more morphological abnormalities than those from patients with moderate teratozoospermia (6% to 10% normal forms). In severely teratozoospermic patients, significantly fewer partially zona-dissected than subzonally inserted embryos implanted. CONCLUSIONS: The decision of which micromanipulation method to perform can possibly be based on careful analysis of sperm morphology.

Coculture of human zygotes on fetal bovine uterine fibroblasts: embryonic morphology and implantation.

Zygotes from in vitro fertilization patients (n = 116) were randomly allocated to culture in either conventional plastic petri dishes or coculture on a monolayer of fetal bovine uterine fibroblasts. Embryos (n = 288) remained 26 to 32 hours in these culture systems. Video tape recording for later morphological analysis (11 parameters) was performed on 117 conventionally cultured and 104 cocultured embryos, shortly before replacement, by an independent observer, unaware of the culture conditions for each embryo. A significantly greater number of cocultured embryos (52%) had "good" morphology (zero or only one abnormal characteristic) as compared with conventionally cultured embryos (30%). The most outstanding morphological characteristic of cocultured embryos was the expanded appearance of their blastomeres. The incidence of implantation per embryo increased from 13% to 19% when the coculture rather than conventional culture system was used, and the incidence of ongoing pregnancy per patient after coculture doubled to 35%.

Blastocyst formation and hatching in vitro following zona drilling of mouse and human embryos.

Hatching in vitro was studied following zona drilling of 507 two-cell mouse embryos using three methods: 1) acidic Tyrode's (AT), 2) partial zona dissection (PZD) using a sharp microneedle, and 3) zona chiseling (CH), using a large beveled needle. PZD and CH were performed while the embryos were kept in a sucrose/PBS solution. Hatching was compared to 191 unmicromanipulated controls. The incidences of cavitation and completion of hatching did not differ between groups, however more micromanipulated embryos (20-25%) hatched partially than controls (9%). The zona pellucida thinned in 59/59 (100%) control blastocysts during expansion, but in only 3/205 (2%) micromanipulated blastocysts. The hatching gap was wide in all control embryos, but smaller in 96/129 (75%) micromanipulated embryos. Partially hatched blastocysts with a "figure-8" shape were found in 59/129 (46%) micromanipulated embryos and in none of the 39 hatching controls. Hatching usually occurred a day earlier in micromanipulated embryos as 214/218 (98%) had started extruding on day 5 as compared to 20/59 (27%) control blastocysts. Fifty percent of 1-day-old human oocytes were fertilized following PZD and reinsemination and 15/31 (48%) were monospermic. Thirteen monospermic embryos cleaved, six compacted and four cavitated--of these, three extruded through the PZD incision upon expansion. The zonae did not thin and one blastocyst twinned spontaneously as it was caught between the thick ridges of the PZD hole. Results indicate that the hatching process is abnormal following zona drilling; more embryos start hatching, extrusion occurs earlier, and many become trapped which may lead to artificial twinning or the formation of trophoblastic vesicles.

A preferential site for sperm-egg fusion in mammals.

The tendency of mammalian sperm-egg fusion to occur at a site away from the first polar body was investigated in a homologous (mouse oocytes and mouse spermatozoa) and in a heterologous model (hamster oocytes and mouse spermatozoa). Following micromanipulation of the zona pellucida either in proximity to or opposite the first polar body, in vitro fertilization was performed and subsequent differences in sperm-egg interaction were evaluated. Since spermatozoa from random-bred mice do not readily penetrate intact zonae pellucidae in vitro, it is likely that zona penetration occurred through the artificial holes in both models. The creation of a gap in the zona pellucida opposite the first polar body was associated with levels of sperm fusion that were significantly higher than those resulting from manipulation near the first polar body. Spermatozoa were rarely found to penetrate the hole completely, and in general few spermatozoa were observed in the pervitelline space. The proximity between pronuclei following sperm penetration was correlated with the position of the incision with respect to the polar body. The findings suggest that breaching the zona pellucida for microsurgical fertilization should be performed away from the microvillus-free area of the oocyte.

The fragile X mental retardation protein is a ribonucleoprotein containing both nuclear localization and nuclear export signals.

Fragile X syndrome is a frequent cause of mental retardation resulting from the absence of FMRP, the protein encoded by the FMR1 gene. FMRP is an RNA-binding protein of unknown function which is associated with ribosomes. To gain insight into FMRP function, we performed immunolocalization analysis of FMRP truncation and fusion constructs which revealed a nuclear localization signal (NLS) in the amino terminus of FMRP as well as a nuclear export signal (NES) encoded by exon 14. A 17 amino acid peptide containing the FMRP NES, which closely resembles the NES motifs recently described for HIV-1 Rev and PKI, is sufficient to direct nuclear export of a microinjected protein conjugate. Sucrose gradient analysis shows that FMRP ribosome association is RNA-dependent and FMRP is found in ribonucleoprotein (RNP) particles following EDTA treatment. These data are consistent with nascent FMRP entering the nucleus to assemble into mRNP particles prior to export back into the cytoplasm and suggests that fragile X syndrome may result from altered translation of transcripts which normally bind to FMRP.

Mitochondria in human offspring derived from ooplasmic transplantation.

Ooplasmic transfer from fertile donor oocytes into potentially compromised recipient patient oocytes has led to the birth of nearly 30 babies worldwide. Cytoplasmic transplantation has caused apprehension, since the mixing of human ooplasm from two different maternal sources may generate mitochondrial (mt) heteroplasmy (both recipient and donor mtDNA) in offspring. This investigation traced the mitochondrial donor population both during the ooplasmic transfer technique and in the bloods of two 1 year old children using mtDNA fingerprinting. Donor ooplasm stained for active mitochondria was transferred into recipient ooplasm and the mitochondria were visualized by confocal microscopy after the microinjection procedure and fertilization. Heteroplasmy was found in the blood from each of the children. This report is the first case of human germline genetic modification resulting in normal healthy children.

Subzonal sperm insertion and the frequency of gamete fusion.

The subzonal sperm insertion technique was applied to assess the potential of motile human spermatozoa to form pronuclei. In 184 mature human oocytes, subzonal sperm insertion was used as the primary mode of insemination in cases with abnormal semen analyses. Oocytes (n = 131) that failed to fertilize in vitro in cases with normal semen profiles were also micromanipulated for secondary insemination. The frequency of sperm fusion, expressed as a percentage, was defined as the total number of male pronuclei formed divided by the total number of spermatozoa inserted subzonally. Our results indicate that 37% of spermatozoa from men with normal semen are able to fuse with the oolemma and decondense within the ooplasm, when placed in the perivitelline space of the oocyte. Excluding the oocytes that appeared morphologically abnormal (presence of cytoplasmic inclusions such as refractile bodies within the ooplasm), the frequency of sperm fusion increased to nearly 60%. Moreover, 14% of subzonally inserted spermatozoa from men with abnormal semen analyses demonstrated an ability to form a pronucleus. The incidence of polyspermy was high, ranging from 30 to 80% in the different groups studied. It is therefore concluded that the human oolemma provides little protection against multiple sperm fusion and that the frequency of gamete fusion is unexpectedly high, even when the spermatozoa are derived from infertile men.

Photosynthesis, Morphology, Leaf Anatomy, and Cytogenetics of Hybrids between C(3) and C(3)/C(4)Panicum Species.

The Laxa group of the Panicum genus contains species which have CO(2) exchange and anatomical characteristics intermediate to C(3) and C(4) photosynthetic types (C(3)/C(4)), and also species characterized as C(3). Hybrids were made between two of the C(3)/C(4) species and two C(3) species. Carbon dioxide exchange and morphological, leaf anatomical, and cytogenetic characteristics of F(1) hybrids between Panicum milioides Nees. ex Trin (C(3)/C(4)) and P. laxum Mez. (C(3)), P. spathellosum Doell (C(3)/C(4)) and P. boliviense Hack. (C(3)), and P. spathellosum and P. laxum were studied. There were no consistent differences in apparent photosynthesis, although two of the three hybrids had higher net CO(2) uptake than the C(3) parent. Values of inhibition of apparent photosynthesis by 21% O(2), CO(2) loss in the light, and CO(2) compensation concentration for the hybrids were between those of the parents. All three hybrids showed leaf anatomical traits, especially organelle quantities in the bundle sheath cells, between those of their respective parents. Linear regression of CO(2) compensation concentration on the percentage of mitochondria and chloroplasts in vascular bundle sheaths of the parents and hybrids gave correlation coefficients of -0.94. This suggests that the reduction in CO(2) loss in the C(3)/C(4) species, and to a lesser degree in the F(1) hybrids, was due to development of organelles and perhaps a higher proportion of leaf photorespiration in bundle sheaths. The overall morphology of the hybrids was so different from the parents that they could be described as new taxonomic forms. The chromosomes in the hybrids were mainly unpaired or paired as bivalents indicating possible homology between some parental genomes.

FMRP associates with polyribosomes as an mRNP, and the I304N mutation of severe fragile X syndrome abolishes this association.

Fragile X mental retardation is caused by the lack of FMRP, a selective RNA-binding protein associated with ribosomes. A missense mutation, I304N, has been found to result in an unusually severe phenotype. We show here that normal FMRP associates with elongating polyribosomes via large mRNP particles. Despite normal expression and cytoplasmic mRNA association, the I304N FMRP is incorporated into abnormal mRNP particles that are not associated with polyribosomes. These data indicate that association of FMRP with polyribosomes must be functionally important and imply that the mechanism of the severe phenotype in the I304N patient lies in the sequestration of bound mRNAs in nontranslatable mRNP particles. In the absence of FMRP, these same mRNAs may be partially translated via alternative mRNPs, although perhaps abnormally localized or regulated, resulting in typical fragile X syndrome.

Repeated sperm injection under the zona following initial fertilization failure.

When fertilization fails following micromanipulative under-zona insemination, it is possible to repeat the procedure adding more spermatozoa to achieve fertilization, embryonic development and pregnancy. We report on 18 human in-vitro fertilization cycles where this approach was used. In nine cycles only late-fertilized embryos were available for transfer, and these gave rise to two viable pregnancies (22.2% per transfer). In six cycles, where a mixture of late- and timely fertilized embryos were available for transfer, two viable pregnancies arose (33.3% per transfer). In three cycles no fertilization was achieved even after reinsemination by repeated under-zona insemination.

Microsurgical fertilization procedures: the absence of stringent criteria for patient selection.

Subzonal sperm insertion and partial zona dissection were applied in 250 in vitro fertilization cycles in couples (n = 200) with abnormal semen analyses; 61 clinical pregnancies were established (24% per egg retrieval). Patients were selected without using minimal cutoff criteria. The study included patients with 0% normal sperm forms (strict criteria), no motile sperm (but some live cells), and sperm counts which could be assessed only after centrifugation. Patients were categorized into three subsets. Group A (n = 116 cycles) failed to fertilize in a previous cycle. Group B (n = 40) was excluded from IVF due to the severity of sperm profiles, such as a maximum of 2% normal forms. Group C (n = 94) constitutes those patients for whom a standard cycle could possibly result in failure. Monospermic fertilization rates were 18% (A), 19% (B), and 24% (C). The incidences of embryo replacement were 63% (A), 53% (B), and 69% (C). Rates of clinical pregnancy were 22% (A), 23% (B), and 28% (C). The presence of one, two, or three semen abnormalities did not correlate with the outcome of microsurgical fertilization. Twenty-two percent of patients with combined oligoasthenoteratozoospermia became pregnant. Moreover, ongoing pregnancies were established in instances with 0% normal sperm forms and no progressively motile spermatozoa. It is concluded that stringent cutoff criteria may not be necessary when both partial zona dissection and subzonal sperm insertion are performed efficiently.

Non-disjunction in human sperm: results of fluorescence in situ hybridization studies using two and three probes.

Fluorescence in situ hybridization using two or three probes was utilized to estimate the incidence of diploidy, the incidence of disomy for the sex chromosomes and chromosomes 16 and 18, and the proportion of Y- and X-chromosome bearing sperm, in a series of normal males. Our results demonstrate the importance of using an approach capable of distinguishing disomy from diploidy, as most donors had levels of diploidy higher than the disomy levels of individual chromosomes. Our analyses suggest the existence of chromosome-specific mechanisms of paternal non-disjunction, as sex chromosome disomy was approximately 1.5 times as common as disomy 16, and over two times as common as disomy 18. In studies of gametic sex ratio, we found little evidence for marked deviation from an expected 1:1 ratio.

Evaluation of Vero cell co-culture system for mouse embryos in various media.

Our objective was to evaluate and compare the efficacy and mechanisms of co-culturing mouse embryos with Vero cells in both Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12) and human tubal fluid (HTF) culture medium. Two-cell CB6F1 mouse embryos were cultured either in the presence of Vero cells (group A) or in culture medium alone (group B). In DMEM/F12 significantly more morulae developed in group A than in group B on day 3 (91 versus 23%; P less than 0.01). In contrast, the mouse embryos grew rapidly in HTF and significant differences were noted only in later embryonic stages (on day 5; 86% and 50%, P less than 0.01 of group A and B respectively, hatching or hatched). Similar experiments using DF1 and ICR mouse strains also revealed enhanced embryo development in the presence of Vero cells. To determine whether the embryo-enhancing effects of Vero cells were due to the removal of toxins or to the secretion of embryotrophic factors, ICR mouse embryos were cultured in fresh media with cells (group A), without cells (group B) and in cell-free conditions using cell-conditioned media which were obtained in the presence (group C) or absence (group D) of embryos. These results demonstrated that completion of hatching was highest (52%; P less than 0.01) in group A after 6 days in culture. There were no significant differences between groups B, C and D (rates of total hatching 18, 17 and 17%, respectively). It is concluded that Vero cells improve the development of mouse embryos and this is likely to be due to removal of substances inhibitory to development.(ABSTRACT TRUNCATED AT 250 WORDS)