Gene expression confirms a potentially receptive endometrium identified by histology in fertile women.

BACKGROUND: To use contemporary biochemical markers to characterize mRNA/gene expression in the potentially fertile secretory endometrium to confirm its identification based on histological characteristics in order to develop a clinically applicable test. METHODS: Nine, fertile, cycling Caucasian women were sampled from one IVF clinic. Endometrial samples were collected from them in two to four menstrual cycles at 2 and 7 days post first significant rise in blood LH. Separate endometrial glands and stroma populations were obtained by laser microdissection. Linear polymerase chain reaction amplified mRNAs which were hybridized to both Affymetrix U133 Plus2 and Agilent 4 × 44K microarrays followed by gene set analysis. Four histopathologists reviewed the sample set using the same histological criteria to date and characterize the non-receptive and potentially receptive samples. RESULTS: mRNA expression of microdissected glands and stroma provided molecular signatures that characterized the two specific phases of the cycle with distinct clustering patterns. Cell proliferation and five other associated biological pathways were significantly down-regulated when the endometrium is considered potentially receptive accompanied by an increase in secreted glycoproteins mRNAs in the potentially receptive glands. Reported histological findings identified the presence of one histological feature characteristic of each phase: glandular mitoses indicated a non-receptive endometrium, whereas a potentially receptive endometrium was distinguished by supranuclear vacuolation. CONCLUSIONS: This study defined a transcriptome characteristic of active cell proliferation in the non-receptive samples with a marked overall down-regulation of this pathway in potentially receptive samples-suggesting a transitional state associated with receptivity but not implantation. However, microarrays involve expensive, specialized testing and require significant post-data analysis. Sampling according to endocrinological and molecular prediction improved the consistency of histological assessment and allowed reliable histological markers of glandular mitosis in the non-receptive phase and supranuclear vacuolation of the potentially receptive endometrium to be identified. Thus, histology can provide an affordable, clinically applicable test in the context of reproduction.

Purification and properties of xanthine dehydrogenase from Pseudomonas acidovorans.

Xanthine dehydrogenase (EC 1.2.1.37) from Pseudomonas acidovorans has been purified to near homogeneity (approx. 65-fold). The enzyme has a molecular weight of about 275 000. Electrophoresis in gels containing sodium dodecyl sulphate showed the presence of two types of subunit with molecular weights of about 81 000 and 63 000. Thus the intact molecule probably contains two of each type of subunit. Xanthine and hypoxanthine are good substrates, and NAD+ is an effective electron acceptor. With xanthine and NAD+ as substrates the purified enzyme has a specific activity of about 20 mumol NADH formed/min per mg protein. Michaelis constants for xanthine and NAD+ are 0.07 and 0.12 mM, respectively, and for hypoxanthine and NAD+ 0.29 and 0.16 mM, respectively.

Purine degradation in Pseudomonas aeruginosa and Pseudomonas testosteroni.

1. Adenine, hypoxanthine, xanthine and guanine are broken down in Pseudomonas aeruginosa and Pseudomonas testosteroni to allantoin by the concerted action of the enzymes adenine deaminase, guanine deaminase, NAD+-dependent xanthine dehydrogenase and uricase. 2. Uric acid is broken down by an unstable, membrane-bound uricase with an unusually low pH optimum. 3. In both strains adenine inhibits growth and xanthine dehydrogenase. A second type of inhibition is manifest only in Ps. testosteroni and concerns the regulation of the biosynthesis of amino acids of the aspartate family. Enzymic studies showed that in this strain aspartate kinase is inhibited by AMP.

Concentration and relationship to progesterone of progesterone-binding globulin in pregnant guinea-pigs: measurement by progesterone tracer binding assay.

A single-point progesterone tracer binding assay (TBA) was developed to measure the concentration of progesterone-binding globulin (PBG) in the serum of pregnant guinea-pigs. Tritiated progesterone was added to dilutions of a reference serum, the percentage bound calculated and a standard curve constructed. The amount of progesterone tracer bound to serum samples was determined, and the concentration of binding protein in the sample relative to the reference was calculated. The molar concentration in the reference serum was determined by Scatchard analysis. The TBA could readily process 30 samples per day. There was a rapid rise in PBG concentrations between days 15 and 20 of gestation, in parallel with progesterone concentrations. The molar ratio of PBG: progesterone was approximately 10:1 after this time, until about day 45 when there was an increase in ratio.

Estrogen-induced transcortin increase and progesterone and cortisol interactions: implications from pregnancy studies.

The concentrations of progesterone, cortisol, estradiol, and transcortin binding capacity (TBC) were measured in plasma samples of women during normal pregnancy. Between 10 weeks and 20 weeks gestation, the mean of TBC increased linearly, and the mean increase in TBC for a given estradiol increment was constant until the estradiol concentrations reached approximately 30 nmol per liter. The results were consistent with the increase in TBC having been induced by estradiol; however, there was an inherent upper limit of response. Progesterone and cortisol were each linearly related to TBC, but the ratios of progesterone:TBC and cortisol:TBC showed no systematic trend throughout the period studied, and there was no systematic relationship between TBC and the progesterone:cortisol ratio. There was, however, a linear relationship between TBC and the progesterone + cortisol sum, such that a unit increase in TBC was accompanied by an approximate unit increase in the total concentration of the two main transcortin binding steroids. Some cases of spontaneous abortion or habitual abortion might be due to aberrant metabolic influence on progesterone of binding protein; in the instances studied, no evidence was found for this.

Nutrition in pregnancy in the Wellington region.

The diets of 115 pregnant women in the Wellington region were assessed for nutrient intake using 24 h dietary recall. Assessment was performed in both the second and third trimester. Women came from three ethnic groups, European (61), Maori (29) and Pacific Islanders (25). Comparisons of nutrient intake were made between these groups. The overall energy intake was similar between the groups (range 4.8-19.7 MJ/d) but Maori (p less than 0.05) and Pacific Islanders (p less than 0.02) had a significant decrease in energy intake from second to third trimester. Pacific Islanders consumed significantly more starch (121 g/d, p less than 0.05) whereas Maori women consumed significantly more sucrose (86 g/d, p = 0.0002). The mean intake in Pacific Islanders contained significantly less calcium (882 mg/d, p = 0.0002) and zinc (9.0 mg/d, p = 0.014). Forty-four percent Europeans, 28% Maori and 51% Pacific Islanders had an estimated iron intake below the minimum safe intake for pregnancy. However dietary iron intake did not relate to the presence of anaemia nor whether iron supplements were given.

Frequency of microdeletions in the azoospermia factor region of the Y-chromosome of New Zealand men.

AIM: To determine the frequency of microdeletions in the azoospermic factor (AZF) genes on the Y-chromosome of New Zealand men attending the Fertility Centre. METHODS: World Health Organisation criteria were used to classify men as normospermic, oligozoospermic, severely oligozoospermic, and azoospermic. Microdeletions were detected from DNA of semen samples by the sequence-tagged site polymerase chain reaction. RESULTS: Microdeletions were detected in 20% (3/15) of azoospermic men, 4% (2/50) of severely oligozoospermic men, 3.2% (2/62) of oligozoospermic men, and 0.7% (1/141) normospermic men. One azoospermic man had multiple non-contiguous deletions. Overall, 5.5% of infertile men had at least one microdeletion in the long arm of the Y-chromosome. One severely oligozoospermic man and one oligozoospermic man had produced unassisted pregnancies. CONCLUSION: New Zealand men attending a Christchurch fertility centre have a similar frequency of microdeletions in the Y-chromosome to other populations. Azoospermic men have a higher frequency of microdeletions than men with less severe spermatogenic failure. Men with microdeletions can have reduced fertility, but are not necessarily sterile.

Down syndrome in monochorionic twins.

Down syndrome in monochorionic twins has rarely been reported. We report such a case diagnosed prenatally. Maternal serum screening was performed at 15 weeks for a twin pregnancy which indicated a risk of greater than 1:50 for Down syndrome. The review of early ultrasonography confirmed monochorionic twins. Amniocentesis at 17 weeks' gestation was performed on one of the twin sacs, which confirmed Down syndrome. A screening scan at 19 weeks' gestation showed isolated absent nasal bones in both twins. Termination of pregnancy was performed subsequently.

Molecular analysis of polymerase gamma gene and mitochondrial polymorphism in fertile and subfertile men.

CAG trinucleotide repeat length in the nuclear polymerase gamma gene (POLgamma) has been shown to be associated with men with reduced fertility. The present study investigated the frequency of CAG repeat length genotypes and three exonuclease motifs of the POLgamma in relation to the frequency of mitochondrial nucleotide substitutions. DNA from semen samples of 93 normozoospermic men and 192 non-normozoospermic men was isolated and the specific regions of the genes were amplified by polymerase chain reactions (PCR) and sequenced to identify mutations. The genotypic frequencies of pooled POLgamma CAG repeat lengths, =10/ not equal 10 heterozygotes and not equal 10/ not equal 10 homozygotes, were significantly different between normozoospermic and non-normozoospermic men (p < 0.05), with non-normozoospermic men having a slightly higher frequency of the =10/=10 genotypes. The allelic frequency for =10 is 0.79 and not equal10 is 0.21 for normozoospermic men and 0.85 and 0.15, respectively, for non-normozoospermic men (p < 0.025). There was no mutation detected in the exonuclease motifs in all the samples tested. Eighty normozoospermic and 124 non-normozoospermic semen samples were analysed for nucleotide substitutions in mitochondrial genes by PCR and sequencing. Heteroplasmic mutations were found in one azoospermic man, four asthenozoospermic men and two normozoospermic men. Only one asthenozoospermic man was heterozygous for the POLgamma genotype. Of the 17 men with non-synonymous nucleotide substitutions, 14 were homozygous for the POLgamma genotype. Non-normozoospermic men had twice as many nucleotide substitutions than normozoospermic men. However, there were no significant differences in the frequencies of nucleotide substitution and POLgamma genotypes in the two groups of men.

Effect of prostaglandin F2 alpha at different stages of guinea-pig pregnancy.

It is believed that suppression of the processes by which prostaglandin F2 alpha is released from the uterus during the estrous cycle is vital to maintenance of pregnancies in guinea-pigs. Prostaglandin F2 alpha was injected into pregnant guinea-pigs at four different stages of gestation to investigate the effect increased prostaglandin might have. The study revealed an alteration in the sensitivity of the pregnancy to prostaglandin F2 alpha as pregnancy progressed. Recovery from the prostaglandin insult was more likely if the injection was given after Day 24 than before Day 18. In some animals the serum progesterone levels fell following the injection and then subsequently recovered. It appears that effects which are potentially hazardous to the pregnancy are countered in a variety of ways.

Adenine phosphoribosyltransferase in Mycoplasma mycoides and Escherichia coli.

Some kinetic properties of the adenine phosphoribosyltransferases from Escherichia coli and Mycoplasma mycoides have been studied. For the E. coli enzyme, Michaelis constants for adenine and 5-phosphoribosyl-1-pyrophosphate (PRPP) are 1.3 and 10 mum, respectively. Adenosine monophosphate, the most effective nucleotide inhibitor, inhibits competitively with respect to PRPP, the inhibition constant being 26 mum. The M. mycoides enzyme has more complex kinetics. The response to increasing PRPP concentration is sigmoidal, the degree of sigmoidality depending on both the concentration of adenine and the pH. At low PRPP levels, high concentrations of adenine are inhibitory. Guanosine monophosphate is the most effective inhibitor, being inhibitory at all pH values, but other nucleotides have been found to activate at pH 7 and inhibit at pH 8. The elution profile of the M. mycoides enzyme from Sephadex suggests an association of enzyme subunits in the presence of PRPP. This is consistent with the observed kinetics if the associated form has increased stability and activity.

Enzymes of purine metabolism in Mycoplasma mycoides subsp. mycoides.

The major pathways of ribonucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides were proposed previously from studies of its usage of radioactive purines and pyrimidines. To interpret more fully the pattern of purine usage, we have assayed cell-free extracts of this organism for several enzymes associated with the salvage synthesis of purine nucleotides. M. mycoides possessed phosphoribosyltransferases for adenine, guanine, and hypoxanthine, purine nucleoside phosphorylase, GMP reductase, GMP kinase, adenylosuccinate synthetase, and adenylosuccinate lyase. Purine nucleoside kinase and adenosine deaminase were not detected. Examination of kinetic properties and regulation of some of the above enzymes revealed differences between M. mycoides and Escherichia coli. Most notable of these were the greater susceptibility of the enzymes from M. mycoides to inhibition by nucleotides and the more widespread involvement of GMP as an inhibitor. Observations on enzyme activities in vitro allow an adequate explanation of the capacity of guanine to provide M. mycoides with its full requirement for purine nucleotides.

Progesterone-binding globulin and progesterone in guinea-pigs after ovariectomy, abortion and parturition.

The level of progesterone in the serum of pregnant guinea-pigs which continued their pregnancies after ovariectomy on Day 30 fell and then rose again. In contrast, progesterone-binding globulin (PBG) was apparently not affected. PBG did however fall in guinea-pigs which aborted and also after parturition. It is apparent therefore that high PBG levels were associated with continued pregnancy. The half life of PBG was found to be approximately 2 days.

Effect of oestrogen on pregnancy of guinea-pigs.

Pregnant guinea-pigs were injected with oestrogen at different stages of gestation. After injections on Days 17 and 18 and on Days 25, 26, 32 and 33 peripheral maternal progesterone concentrations were lower than in control animals. Histology showed deleterious changes in the corpora lutea. After injections on Days 10 and 11 the maternal progesterone concentrations remained in the normal range for some time in 12 of 16 guinea-pigs and the corpora lutea appeared histologically normal. Oestrogen therefore affects the pregnancy differently at different stages of gestation.

Association of a novel human mtDNA ATPase6 mutation with immature sperm cells.

This study reports the first clearly defined heteroplasmic mutation in immature human sperm cells. The human sperm mitochondrial genome from residue 8186-9341 was analysed with the aim of identifying point mutations which may be associated with human male infertility. The semen samples analysed were obtained from 88 fertile men, 19 with oligozoospermia, and 12 with severe oligozoospermia. Using single strand conformation polymorphism analysis a heteroplasmic T to C transition was detected in the ATPase6 gene, at nucleotide position 8821, in semen samples from one out of 12 (8%) severely oligozoospermic men, but not in oligozoospermic men or normospermic men. This mutation changed the amino acid serine to proline at residue 99 of the mitochondrial ATPase6 in a region which is highly conserved in other vertebrates including rat, bovine, chicken, salmonids and Xenopus. The mutation was detected in semen samples collected from the same man 9 months apart and in peripheral blood lymphocytes. Single sperm cell analyses did not find this mutation in the mature sperm, but the mutation was detected in 7% of immature spermatids. Our finding suggests that immature spermatids with this mutation fail to develop fully.

Electroporation of salmon sperm for gene transfer: efficiency, reliability, and fate of transgene.

Uptake of exogenous DNA by electroporated salmon sperm for gene transfer is being investigated. Our studies show that electroporated salmon sperm cells were more efficient and more reliable than untreated sperm in picking up exogenous DNA and subsequently transferring the DNA into salmon embryos. Indirect evidence suggest that some of the exogenous DNA was internalized in the sperm nuclei. The taken up DNA retained its integrity as demonstrated by PCR. The foreign DNA was detected in 15-month-old fish, and had a mosaic pattern of distribution. Integration of the foreign DNA occurred infrequently, and the expression of the foreign genes was poor. The potential of sperm-mediated gene transfer as a routine protocol for mass gene transfer in salmon will be dependent on the improvement of integration and expression of the foreign gene.

High incidence of single nucleotide substitutions in the mitochondrial genome is associated with poor semen parameters in men.

Single nucleotide polymorphisms (SNPs) in 7000 bp of the mitochondrial genome, encompassing 15 coding regions from COI to ND5, were characterized by single strand polymorphism analysis and confirmed by DNA sequencing. About 2.4% of normozoospermic men and 8.4% of men with poor semen quality had at least one nucleotide substitution. Most of the substitutions occurred in the third codon and did not change the amino acid. Hydrophobicity plots of the proteins with changes in an amino acid as a result of a nucleotide substitution suggested that they did not affect the function of the protein. The two most common substitutions at nucleotide (nt) 9055 and 11719 had significantly higher frequencies in men with reduced sperm motility. Eleven percent of the men with poor semen parameters and 1.3% of normozoospermic men had a 9055 substitution, 12% of the men with poor semen parameters had a substitution at nt 11719, but none of the normozoospermic men had this substitution. All the patients with these substitutions had reduced sperm motility and/or low sperm count. These SNPs in the mitochondrial genome were in a homoplasmic state. Thus, we propose that possessing these mitochondrial mutations compromises the semen quality of these men.