Prolonging the interval from ovarian hyperstimulation to laparoscopic ovum pick-up improves oocyte yield, quality, and developmental competence in goats.

The objective was to evaluate the effect of the interval between ovarian hyperstimulation and laparoscopic ovum pick-up (LOPU) on quality and developmental competence of goat oocytes before and after in vitro maturation (IVM) and intracytoplasmic sperm injection (ICSI). Estrus was synchronized with an intravaginal insert containing 0.3g progesterone (CIDR) for 10d, combined with a luteolytic treatment of 125 microg cloprostenol 36 h prior to CIDR removal. Ovaries were hyperstimulated with 70 mg FSH and 500 IU hCG given im 36, 60, or 72 h prior to LOPU (n=15, 16, and 7 does, respectively). For these groups, oocyte retrieval rates (mean+/-S.E.M.) were 24.7+/-2.9, 54.5+/-4.7, and 82.8+/-4.6% (P<0.001), and the proportions of cumulus-oocyte complexes (COC) with more than five layers of cumulus cells were 29.7+/-8.3, 37.6+/-6.9, and 37.3+/-7.0% (P<0.001). The proportion of IVM oocytes was highest at 72 h (82.1+/-2.8%; P<0.05), with no significant difference between 36 and 60 h (57.3+/-8.9% and 69.0+/-8.4%). Cleavage rates of ICSI embryos were 4.2+/-4.2, 70.9+/-8.4, and 78.9+/-8.2% with LOPU 36, 60, and 72 h post FSH/hCG (P<0.01), with a lower proportion of Grade-A embryos (P<0.05) following LOPU at 36 h compared to 60 and 72 h (29.7+/-8.3%, 37.6+/-6.9%, and 37.3+/-7.0%). In summary, a prolonged interval from FSH/hCG to LOPU improved oocyte retrieval rate and oocyte quality. Therefore, under the present conditions, LOPU 60 or 72 h after FSH/hCG optimized yields of good-quality oocytes for IVM and embryo production in goats.

Somatic cell nuclear transfer using transported in vitro-matured oocytes in cynomolgus monkey.

Somatic cell nuclear transfer (SCNT) is not successful so far in non-human primates. The objective of this study was to investigate the effects of stimulation cycles (first and repeat) on oocyte retrieval and in vitro maturation (IVM) and to evaluate the effects of stimulation cycles and donor cell type (cumulus and fetal skin fibroblasts) on efficiency of SCNT with transported IVM oocytes. In this study, 369 immature oocytes were collected laparoscopically at 24 h following human chorionic gonadotrophin (hCG) treatment from 12 cynomolgus macaque (Macaca fascicularis) in 24 stimulation cycles, and shipped in pre-equilibrated IVM medium for a 5 h journey, placed in a dry portable incubator (37 degrees C) without CO(2) supplement. A total of 70.6% (247/350) of immature oocytes reached metaphase II (MII) stage at 36 h after hCG administration, MII spindle could be seen clearly in 80.6% (104/129) of matured IVM oocytes under polarized microscopy. A total of 50.0% (37/74) of reconstructive SCNT embryos cleaved after activation; after cleavage, 37.8% (14/37) developed to the 8-cell stage and 8.1% (3/37) developed to morula, but unfortunately none developed to the blastocyst stage. Many more oocytes could be retrieved per cycle from monkeys in the first cycle than in repeated cycles (19.1 vs. 11.7, p < 0.05). There were no significant differences in the maturation rate (70.0 vs. 71.4%, p > 0.05) and MII spindle rate under polarized microscopy (76.4 vs. 86.0%, p > 0.05) between the first and repeat cycles. There were also no significant differences in the cleavage rate, and the 4-cell, 8-cell and morula development rate of SCNT embryos between the first and repeat cycles. When fibroblast cells and cumulus cells were used as the donor cells for SCNT, first cleavage rate was not significantly different, but 4-cell (50.0 vs. 88.9%, p < 0.05) and 8-cell (0 vs. 51.9%, p < 0.01) development rate were significantly lower for the former. In conclusion, the number of stimulation cycles has a significant effect on oocyte retrieval, but has no effect on maturation and SCNT embryo development; however, different donor cell types (cumulus and fibroblast) resulted in different developmental potentials of SCNT embryos.

Therapeutic and reproductive cloning--implications and recommendations.

The announcement of Dolly's birth took the world by storm, mainly because what was thought impossible has become possible. Optimism that new approaches in agriculture and medicine abound, as much as fear and imagination leading to Frankenstein-like scenarios. Scientifically, cloning refers to replicating an animal with the same nuclear genetic material; whilst it may refer to embryos as the source material, the current storm refers to differentiated ("somatic") cells. Cloning technology is useful in the following areas: agriculture, to produce animals of superior or specific qualities; endangered animals, to increase genetic diversity through widening the gene pool; understanding fundamental questions in developmental embryology, through the use of laboratory animals; and in human therapy, to produce cells and possibly tissues for repair and regeneration. In the first 2 instances, it is reproductive cloning. In the last instance, it is therapeutic cloning, as no individuals are "produced". Human reproductive cloning is not allowed by all governments that have deliberated on it, and therapeutic cloning is allowed by some under certain circumstances. As therapeutic cloning has great potential in cures of many diseases, it should be allowed but with safeguards to prevent abuse and reproductive cloning in the human.

Prognostic value of Y deletion analysis: How reliable is the outcome of Y deletion analysis in providing a sound prognosis?

Y chromosomal microdeletions at the azoospermia factor (AZF) locus have been implicated as one of the major causes of idiopathic male infertility. The availability of intracytoplasmic sperm injection (ICSI) in treating a variety of male infertility has raised the risk of the transmission of Y microdeletions from father to son. In many IVF centres, Y microdeletion analysis has been used as a diagnostic tool for genetic counselling of infertile couples. Presently, the only prognosis that can be derived from Y microdeletion analysis is that the affected male offspring would benefit from proper clinical management of their infertility. Prognoses based on the pattern of Y microdeletions in relation to phenotype are rather subjective and inconclusive because of insufficient data to derive a definitive correlation whose significance can be determined by statistical analysis. Standardization of the number and choice of sequence-tagged sites (STS), whose deletions result in defective spermatogenesis, for the polymerase chain reaction (PCR) analysis of Y microdeletions would enhance its reliability in the interpretation of the results which is crucial for therapeutic decision-making. Furthermore, in-depth understanding of the gene functions in male infertility, especially at the AZF locus, would contribute greatly to the quality of the prognostic value of Y microdeletion analysis.

Mutations in the promoter region of the androgen receptor gene are not common in males with idiopathic infertility.

Molecular studies on the role of the androgen receptor in male infertility have thus far concentrated solely on exonic regions of the androgen receptor gene. We have therefore screened for the first time the androgen receptor gene 5' untranslated region (nucleotides -153 to +237 ) in 240 males with idiopathic infertility for lesions which could potentially impair spermatogenesis. This region encompasses the androgen receptor gene promoter. DNA was extracted from blood leukocytes and the polymerase chain reaction was used to amplify the promoter region as two overlapping products. Single strand conformational polymorphism analysis was carried out on these products to screen for mutations. This analysis did not reveal the presence of any gross deletions or mutations. Our results thus preclude aberrations in the promoter region of the androgen receptor gene as a common factor in the aetiology of idiopathic male infertility.

Y chromosome microdeletions, in azoospermic or near-azoospermic subjects, are located in the AZFc (DAZ) subregion.

Submicroscopic deletions of the Y chromosome and polymorphisms of the androgen receptor (AR) gene in the X chromosome have been observed in men with defective spermatogenesis. To further define the subregions/genes in the Y chromosome causing male infertility and its relationship to polymorphisms of the AR polyglutamine tract, we screened the genomic DNA of 202 subfertile males and 101 healthy fertile controls of predominantly Chinese ethnic origin. Y microdeletions were examined with 16 sequence-tagged site (STS) probes, including the RBM and DAZ genes, spanning the AZFb and AZFc subregions of Yq11, and related to the size of trinucleotide repeat encoding the AR polyglutamine tract. Y microdeletions were detected and confirmed in three out of 44 (6.8%) of azoospermic and three out of 86 (3.5%) severely oligozoospermic patients. No deletions were detected in any of the patients with sperm counts of >0.5 x 10(6)/ml, nor in any of the 101 fertile controls. All six affected patients had almost contiguous Y microdeletions spanning the entire AZFc region including the DAZ gene. The AZFb region, containing the RBM1 gene, was intact in five of the six subjects. Y deletions were not found in those with long AR polyglutamine tracts. Our study, the first in a Chinese population, suggest a cause and effect relationship between Y microdeletions in the AZFc region (possibly DAZ), and azoospermia or near-azoospermia. Y microdeletions and long AR polyglutamine tracts appear to be independent contributors to male infertility.

Fertilization, embryonic development and implantation of mouse oocytes with one or two laser-drilled holes in the zona, and inseminated at different sperm concentrations.

The zonae pellucidae of mouse oocytes were photo-ablated by an ultraviolet laser (337.1 nm) to create one or two 10 microm holes. The pulse energy used was approximately 3 microJ/s, with a frequency of 10 pulse/s. These laser zona-drilled (LZD) oocytes with one hole (LZD1) or two holes (LZD2) and the zona-intact controls were inseminated with spermatozoa at standard concentrations of 5x10(4), 5x10(5), 1x10(6) and 2x10(6)/ml. Fertilization was significantly improved at all sperm concentrations in LZD1 and LZD2 oocytes as compared to the controls. However, there was no significant difference in fertilization rates between LZD1 and LZD2. LZD2 produced significantly higher numbers of blastocysts at day 5. Hatching was significantly enhanced in the presence of either one or two holes in the zona. Polyploidy was generally absent, except in LZD2 oocytes (1%) inseminated at higher sperm concentrations. Differential cell counts of expanded LZD blastocysts were similar to those of the controls. Significantly fewer LZD2 blastocysts implanted and produced viable fetuses than LZD1 and control blastocysts. Morphological abnormalities of the fetuses were absent in all three groups. Laser zona-drilling using the ultraviolet laser was shown to be fast, efficient and safe.

Intracytoplasmic sperm injection of mouse oocytes with 5 mM Ca2+ at different intervals.

The objective of this investigation was to determine whether intracytoplasmic sperm injection (ICSI) can be performed in the mouse. Metaphase II oocytes were obtained from F1 hybrid mice (C57BL x CBA) by i.p. injections of 10 IU pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG) administered 48 h apart. Oocytes with cumulus oophorus were retrieved 13-14 h post HCG. Cumulus was dispersed with 0.1% hyaluronidase. Mouse spermatozoa were obtained from the cauda epididymides of males of the same strain. The spermatozoa were processed by the standard swim-up procedure. The harvested spermatozoa were then incubated for 1.5 h to allow capacitation. Healthy oocytes were injected with 3-4 pl 5 mM Ca2+, followed by one live morphologically normal spermatozoon into the cytoplasm at intervals of 0, 0.5, 1, 2 and 3 h. The proportion of 2-cell embryos that developed from oocytes injected with Ca2+ and spermatozoa ranged between 29.5 and 36.5% in all groups, with no statistical difference between treatments. Chromosomal analysis showed that two-thirds of the ICSI-derived 2-cell embryos were diploid. The proportion of parthenogenetically activated embryos in the ICSI groups was similar to that in the control group (8-10%) which was injected with Ca2+ and polyvinyl pyrrolidone only. The proportion of blastocysts that developed in culture from the ICSI-derived 2-cell embryos was of the order of 36-42%. Some blastocysts were used for cell number counts. There was a significant increase in total and inner cell mass counts of blastocysts in which the spermatozoon was injected at 2 and 3 h following Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Early sperm-egg interaction after sperm microinjection.

The early events of sperm-egg interaction occurring 1-3 h after multiple sperm injection into the perivitelline space (PVS) and after direct injection into the ooplasm of human oocytes are reported. The sperm acrosome reaction occurred in the PVS but was not detected within the ooplasm. Sperm in the PVS were incorporated into the ooplasm in the usual manner described in vitro, after completion of the acrosome reaction, and a block to polyspermy was evident at the oolemma. However, sperm incorporation into the ooplasm was not clearly defined and requires further investigation. Sperm were also incorporated into oolemma-bound vacuoles within the ooplasm and breaches in the ooplasm were seen after intracytoplasmic injection. Both normal and abnormal sperm were found in the PVS and ooplasm, even though sperm from donors with normal semen parameters were used. The effect of freezing in liquid nitrogen on demembranation of sperm for microinjection is also reported.

In vitro decondensation of mammalian sperm and subsequent formation of pronuclei-like structures for micromanipulation.

In this study, we describe an efficient protocol for the formation of in vitro developed pronuclei for micromanipulation techniques. Our approach involved incubation of demembranated or permeabilized mammalian sperm in a phosphate buffer supplemented with heparin and beta-mercaptoethanol. Under the prevailing conditions, we achieved a uniform and reliable synchronous decondensation of sperm nuclear DNA. This initial decondensation facilitated the removal of mammalian protamines upon subsequent incubation in an amphibian egg extract. The interchange of protamines for histones to stabilize the DNA structure is recognized as a prerequisite for pronuclear formation. Furthermore, immunocytochemical studies have revealed that pronuclear development is accompanied by the formation of a nuclear lamina with corresponding DNA synthesis. The method described gave a high yield of nuclei during pronuclear formation. Ultimately, our aim is to transfer the in vitro-developed pronuclei into mammalian oocytes by micromanipulation. This novel procedure may prove useful in alleviating severe male factor problems especially in oligozoospermic cases in our in vitro fertilization center.

Micro-insemination sperm transfer (MIST) and its application to male subfertility: current strategies to improve results.

It can be difficult to achieve a pregnancy for patients with severe male factor subfertility. Hence, micro-manipulation techniques, have been applied to this problem. Direct deposition of sperm into the oocyte under the zona (micro-insemination sperm transfer, MIST) has improved the fertilisation rates, but pregnancy rates have been very low. This paper describes the possible new strategies to improve the technique currently. They include methods to improve sperm recovery, to improve acrosome reaction, to improve the quality of embryos by using co-cultures and to wait for good cleavage before transfer, and to improve luteal phase support. There are also many new techniques being developed which may contribute to further pregnancy successes. They include the use of electro-fusion, laser-fusion and the Xenopus cell-free system.