Parthenogenetic activation of marmoset (Callithrix jacchus) oocytes and the development of marmoset parthenogenones in vitro and in vivo.

Mammalian oocytes can be induced to resume meiosis without fertilization, and the resulting parthenogenetic embryos carry only maternal chromosomes. Human oocytes can be activated by many chemical and physical stimuli, but postimplantation studies of human parthenogenetic embryos are not ethically acceptable. The common marmoset monkey (Callithrix jacchus) is a good model for studying primate parthenogenetic development postimplantation, since follicular aspiration, embryo transfer, and early postimplantation development of biparental embryos have already been described. Marmoset oocytes were either subjected to two series of six electrical pulses (DC; 2 kV/cm and 70 microsec) or were incubated in 7% ethanol in PBS. Ninety-two percent (68 of 74) and 20% (8 of 40) of marmoset oocytes were activated by electrical stimulus or ethanol, respectively. Parthenogenetic (n = 3) or in vitro-fertilized (n = 2) embryos were transferred at the 4-cell stage to synchronized recipient female marmosets (n = 5). Progesterone, chorionic gonadotropin, and inhibin in the peripheral plasma of recipient animals were measured. After 33 days of gestation, recipient animals were perfused and the uteri were collected. The 2 females that had received biparental embryos and 2 of the 3 females that had received parthenogenetic embryos displayed biochemical and histological evidence of implantation. This is the first report that a primate embryo comprising only parthenogenetic cells is capable of implantation. This highlights the need to scrutinize levels of parthenogenesis associated with human assisted reproductive technologies. Marmoset parthenogenones also provide a unique model for elucidating the roles of parental genomes in primate development.

Clinical experience with preimplantation diagnosis of sex by dual fluorescent in situ hybridization.

PURPOSE: Our purpose was to assess the clinical application of dual fluorescent in situ hybridization (FISH) for the diagnosis of sex in the human preimplantation embryo. RESULTS: Over a 2-year period, 18 couples at risk of transmitting X-linked recessive disorders underwent preimplantation diagnosis of embryo sex by dual FISH with X and Y chromosome-specific DNA probes. A total of 27 in vitro fertilization (IVF) treatment cycles led to nine pregnancies; 7 reached the stage of clinical recognition, of which 2 spontaneously aborted. There were five live births, three singleton and two twin: none in disagreement with the diagnosed sex. The diagnosis was corroborated in 51 of the 74 nontransferred embryos. The efficiency of the procedure improved throughout the four treatment cycles. This was reflected in the increased proportion of double embryo transfers (from 50% in series 1 and 2 to 100% in series 3 and 4), with a consequent improvement in pregnancy rate (from 28 to 71% per embryo transfer). The excess of male embryos (male:female, 60:40 overall) and the high proportion of biopsied embryos with abnormal numbers of X and Y chromosome signals (14.5%) effectively reduced the number of normal female embryos available for transfer. CONCLUSION: Dual FISH is an efficient technique for determination of the sex of human preimplantation embryos and the additional ability to detect abnormal chromosome copy numbers, which is not possible via the polymerase chain reaction, (PCR), makes FISH the preferred technique.

In vitro fertilization and embryo development in the marmoset monkey (Callithrix jacchus).

Oocytes aspirated from preovulatory (i.e. > or = 2 mm) follicles of marmoset monkeys were graded for maturity according to the degree of cumulus expansion, grade I being most mature and grade IV least mature. After preincubation for 2-5, 9-11 or 21-29 h, 82% of oocytes could be fertilized using epididymal spermatozoa and only 2.3% were polyspermic. Fertilization rate was lowest (60%) in grade IV oocytes and all oocytes preincubated for 2-5 h (53%). Fertilization rate increased to 92% in oocytes preincubated for 21-29 h. Embryos developed in vitro to a mean of eight cells. Embryo development was unaffected by oocyte maturity but correlated with preincubation time. Oocytes preincubated for 2-5 h developed into embryos with significantly fewer cells than those preincubated for 9-11 or 21-29 h (P < 0.001). Fifty-six per cent of embryos showed delayed cleavage and these had fewer cells than non-delayed embryos (P < 0.001). When oocytes were preincubated for 2-5 h, development of all resulting embryos was delayed. However, only 17 and 58% of embryos developing from oocytes preincubated for 9-11 and 21-29 h, respectively, were delayed and this was independent of oocyte maturity.

Quantitative ultrastructural analysis of sperm tails reveals flagellar defects associated with persistent asthenozoospermia.

Sperm tail morphology was examined in 10 infertile asthenozoospermic men to determine whether poor sperm motility was caused by ultrastructural defects of the flagellum. In this quantitative analysis, the numbers of outer doublet and central pair microtubules, outer and inner dynein arms and radial spokes were counted in transverse sections of 75 axonemes from each patient and compared with similar data previously collected from 10 men with normal semen characteristics. Four patients had axonemal defects: two had severe microtubule abnormalities and two had more subtle but statistically significant deficiencies of dynein arms. These abnormalities would not have been detected by more commonly used qualitative examination. Three patients had no detectable ultrastructural abnormalities of the sperm tail, possibly indicating a metabolic deficiency. A further three patients had mid-piece abnormalities. Two had few, if any, flagellar mitochondria and the third patient had irregular and disorganized mitochondria. Quantitative ultrastructural analysis has revealed axonemal abnormalities in seven of 10 patients with previously unexplained asthenozoospermia.

Dual fluorescent in situ hybridisation for simultaneous detection of X and Y chromosome-specific probes for the sexing of human preimplantation embryonic nuclei.

Dual fluorescent in situ hybridisation has been used for the simultaneous detection of X and Y chromosome-specific probes in single cleavage nuclei from disaggregated 4- to 7-cell human embryos. Based on the presence of a Y signal or 2 X signals in the absence of a Y, 89% of poor quality metaphases and 72% of interphase nuclei could be classified as male or female. With further refinements, this technique will offer a credible alternative to the polymerase chain reaction for the diagnosis of sex in human preimplantation embryos in families segregating for X-linked genetic disease.

Young's syndrome (obstructive azoospermia and chronic sinobronchial infection): a quantitative study of axonemal ultrastructure and function.

The ultrastructure and function of nasal cilia and sperm tails were examined in 23 men with Young's syndrome and compared with data previously collected from 10 normal subjects. Quantitative electron microscopic assessment showed that sperm tails from patients with Young's syndrome contained significantly fewer central pair microtubules, radial spokes, and inner dynein arms, and their cilia contained less inner dynein arms than normal subjects. The Young's syndrome patients had normal in vitro ciliary beat frequency (11.4 +/- 0.9 Hz), and 12 of the 23 had normal nasal mucociliary clearance (15.0 +/- 5.0 minutes). However, the remaining 11 had markedly abnormal nasal mucociliary clearance in vivo. In these patients, the deficiency of ciliary inner dynein arms did not appear to affect ciliary function in vitro but may under mucus loading lead to abnormal in vivo ciliary function. The consistent abnormalities shown in cilia and sperm tails, though apparently minor, constitute a common factor in both the reproductive and respiratory tracts which may, in combination with abnormalities in the in vivo environment, lead to the features of Young's syndrome.

Allocation of cells to the inner cell mass and trophectoderm of 3/4 mouse embryos.

The allocation of cells to the inner cell mass (ICM) and trophectoderm (TE) was investigated at 6-h intervals from 78 h to 102 h after hCG injection in 3/4 mouse embryos to determine the effect of removal of a single blastomere at the 4-cell stage on early differentiation. The procedures used to produce 3/4 embryos had little effect on embryo development. Embryos that had a single blastomere removed and then re-aggregated (RA embryos) had the same total number of cells as untreated (UT) embryos except at 78 h (P less than 0.05) and 102 h (P less than 0.01) post hCG where there were slightly less cells in RA embryos. Three-quarter embryos always had significantly fewer cells than RA embryos (P less than 0.001), with an average of 74% of the total cell number of RA embryos. As expected, 3/4 embryos always had significantly fewer cells in the ICM and TE compared with RA embryos (P less than 0.001). However, the ICM:TE ratio was also significantly lower in 3/4 embryos compared with RA embryos at 84, 96, and 102 h post hCG, indicating that the allocation of cells to the ICM and TE was disturbed. The ICM:TE ratio of 3/4 embryos could not be manipulated if either an early- or late-dividing blastomere was selectively biopsied at the 4-cell stage; this suggests that the known preferential contribution of an early-dividing blastomere to the ICM is not cell autonomous.

Successful single-cell biopsy and cryopreservation of preimplantation mouse embryos.

We have previously observed that preimplantation embryo biopsy in the mouse causes a reduction in implantation rate in utero. After minor modifications to the technique, we now find that sampling a single blastomere from the 4-cell mouse embryo does not compromise continued development in vitro or in vivo. When transferred to pseudopregnant foster mice, 60.3% and 64.3% of biopsied and control embryos, respectively, implanted into the uterine wall, and 52.6% and 52.4% of biopsied and control embryos, respectively, developed into fetuses. In a separate series of experiments, we have demonstrated that biopsied mouse embryos can be successfully cryopreserved by ultrarapid freezing even though they have a punctured zona pellucida. Biopsied (frozen-thawed), control (frozen-thawed), and nonfrozen embryos had an implantation rate of 81.1%, 74.3%, and 74.1%, respectively, and a fetal formation rate of 62.2%, 62.9%, and 66.7%, respectively.

Biopsy of preimplantation mouse embryos: development of micromanipulated embryos and proliferation of single blastomeres in vitro.

We have developed a technique to sample the preimplantation embryo, which may, in the future, be applied to prenatal diagnosis of genetic disease. Using micromanipulation, we aspirated a single blastomere from 4-cell mouse embryos. This procedure had no effect on in vitro development; 98% of control and 94% of biopsied embryos reached the blastocyst stage after 48 h in culture. Furthermore, after transfer to pseudopregnant recipient mice, the rate of fetal development of biopsied embryos was not significantly different from control embryos, although implantation rate was significantly reduced (mean +/- SD: biopsied 53.1 +/- 4.0, control 81.8 +/- 8.4, p less than 0.001). For the first time we have produced monolayer cell cultures derived from single preimplantation blastomeres. Individual biopsied blastomeres were cultured in vitro on different extracellular matrix components. Significantly greater cell proliferation was obtained in wells coated with fibronectin (FN), laminin (LN), and a complex of laminin and nidogen (LNC) than in a less specific matrix of swine skin gelatin (SSG). Mean (+/- SE) cell nuclei number per well after 6 days in culture was 6.4 +/- 2.1, 11.9 +/- 1.5, 19.8 +/- 2.9, and 20.9 +/- 2.6 in wells coated with SSG, LN, FN, and LNC respectively.

Human male infertility caused by degeneration and death of sperm in the epididymis.

Four patients with persistent oligospermia and necrospermia were found to have severely degenerated sperm in the ejaculate. However, in those examined, testicular sperm were ultrastructurally normal, indicating that sperm degeneration and death was occurring during epididymal passage or storage or both or upon mixing with the seminal plasma at ejaculation. Seminal plasma was found to be nontoxic to normal donor sperm. In three patients, frequent ejaculation (two ejaculates per day for 4 or 5 days) was used to deplete epididymal sperm reserves and reduce the period spent in the epididymis. This resulted in a threefold to sevenfold increase in percentage of motile sperm in the ejaculate and a similar increase in sperm motility index. The authors propose the term "epididymal necrospermia" to describe this previously undefined type of male infertility.

Kartagener's syndrome with motile cilia and immotile spermatozoa: axonemal ultrastructure and function.

A 35-yr-old infertile man with chronic sinobronchial disease and dextrocardia (Kartagener's syndrome) was found to have immotile sperm and motile nasal cilia in vitro. Ciliary beat frequency in vitro was normal, but in vivo nasal mucociliary clearance was markedly prolonged. Quantitative electron microscopy demonstrated a severe reduction in spermatozoal outer and inner dynein arms compared with normal (p less than 0.001) but normal numbers of outer doublets, central microtubules, and radial spokes were seen. In 2 samples of nasal cilia collected 14 months apart, the number of inner dynein arms was significantly reduced from normal (p less than 0.001), but normal numbers of radial spokes and microtubule structures were seen. Ciliary outer dynein arms were slightly reduced in 1 specimen (p less than 0.001) but were normal in the other. It is suggested that the reduction in the number of ciliary inner dynein arms does not affect ciliary motility in vitro but that, under the increased load of mucus in vivo, this defect prevents the cilia from functioning normally. The difference in axonemal ultrastructure between cilia and spermatozoa from the same patient further suggests a separate genetic control of their structural components.

Structural heterogeneity of the axonemes of respiratory cilia and sperm flagella in normal men.

The ultrastructure of normal human cilia and flagella was examined and quantitatively assessed to determine the normal variations in the structure of the axoneme. Ciliated respiratory epithelial cells and spermatozoa from 10 normal, nonsmoking male volunteers who had normal semen parameters were fixed for electron microscopy. Tannic acid and MgSO4 were included during fixation to enhance, in particular, axonemal components. In 75 axonemal cross sections per sample, the number of outer doublet and central singlet microtubules, outer and inner dynein arms, and radial spokes were recorded. Statistical analysis of the results showed a marked reduction, from the expected value of nine, in the numbers of inner dynein arms (mean +/- SE, cilia, 5.31 +/- 0.13; sperm, 5.38 +/- 0.16) and radial spokes (cilia, 4.95 +/- 0.22; sperm, 5.80 +/- 0.19). The ideal axoneme with all its structural components was seen in only 0.13% of cilia and 0.80% of sperm tails. Significantly more doublet microtubules (P less than 0.05) and less central microtubules (P less than 0.01) and radial spokes (P less than 0.01) were seen in cilia than in sperm tail axonemes. Between subjects there was little variation in the mean number of a structure seen per axoneme. However, within each sample, the variation was considerably higher, particularly for the inner and outer dynein arms and radial spokes. The doublet microtubules had significantly greater standard deviations in the sperm tails compared with the cilia (P less than 0.01), and furthermore, a significantly greater number of sperm tails compared with cilia showed the incorrect number of doublet microtubules (P less than 0.02). In one semen sample, with normal semen analysis, 20% of the sperm tails showed incorrect numbers of doublet microtubules, ranging from 12 + 2 to 5 + 2 compared with only 1.3% in cilia from this subject. This study has demonstrated that the ideal axoneme is rarely seen even in normal samples, probably because of the technical difficulties in resolution and visualization, and stresses the need for thorough documentation of axonemal ultrastructure. This work provides a normal data base for comparison with patients who have chronic respiratory disease and suspected infertility.

The influence of luteinizing hormone on the Leydig cells of cryptorchid rat testes.

The influence of circulating LH levels on Leydig cells from cryptorchid adult rats was examined after ablation of the pituitary. After 2 weeks cryptorchidism, serum FSH and LH levels rose 2-fold while serum testosterone (T) remained unchanged. Leydig cells were hypertrophied and showed an increased response to in vitro hCG stimulation. Two weeks after hypophysectomy (hypox), serum hormone levels (LH, FSH and T), Leydig cell size, cytoplasm, organelle content and in vitro T production were all dramatically reduced. However, when hypophysectomy was combined with cryptorchidism (hypox/crypt), there was an increase in Leydig cell size, compared to hypophysectomy alone, in the presence of very low levels of serum FSH, LH and T. Compared to the hypophysectomised state, the mitochondria were larger and the cytoplasm contained more smooth endoplasmic reticulum. The response of the hypox/crypt testes to in vitro hCG stimulation, though significantly less than the cryptorchid testes, was significantly greater than the hypox testes. These results demonstrate that the changes observed in the Leydig cell after cryptorchidism can occur in the absence of peripheral pituitary hormones and are consistent with the hypothesis that a local feedback loop exists within the testis.