Fertility after vaginal or uterine deposition of dog semen frozen in a tris extender with or without Equex STM paste

Twenty-five bitches were artificially inseminated with semen that was frozen-thawed using an egg yolk-Tris-glucose-citrate extender containing 5% glycerol with, or without the addition of 0.5% Equex STM Paste. Semen was collected on 2 occasions from 11 dogs, pooled, and evaluated for sperm motility, morphology and plasma membrane integrity. Each pool was then divided in 2 parts, diluted with 1 of the 2 extenders, and frozen in 0.5-mL straws. In the bitches, plasma progesterone was assayed daily during late proestrus and estrus. Artificial insemination (AI) was performed twice on Days 3 and 5 after the estimated LH peak. For each insemination, 200x10(6) spermatozoa were used. Ten bitches were inseminated with semen frozen without Equex: In 5 females, semen was deposited transcervically into the uterus with the aid of a fiberoptic endoscope and a urethral catheter, while the remaining 5 bitches were inseminated in the cranial vagina using a Norwegian catheter. Fifteen bitches were inseminated with semen frozen-thawed with Equex: Two groups of 5 bitches were inseminated according to the techniques described above, while 5 bitches were inseminated vaginally using the Osiris catheter. Pregnancy was diagnosed and the number of fetuses counted by ultrasound examination. Post-thaw, spermatozoa frozen with Equex tended to have higher total and progressive motility and to survive longer in vitro than when the extender without Equex was used. Spermatozoal concentration, age of the bitches, duration of heat and estrus, and progesterone concentration at LH peak and at the first and second AI did not differ among the 5 groups. The overall pregnancy rate of 84% (21/25) was close to what can be expected from well controlled natural matings. For both freezing extenders tested, 5/5 bitches were pregnant after uterine deposition of semen and 4/5 were pregnant when semen was deposited in the anterior vagina using the Norwegian catheter. With the Osiris catheter, 3/5 inseminations resulted in a pregnancy. No significant differences in pregnancy rate or number of fetuses were found between groups, site of deposition or freezing extender.     

Marked inhibition of retinal neovascularization in rats following soluble-flt-1 gene transfer

BACKGROUND: In mouse models of retinopathy of prematurity (ROP) inhibitors of vascular endothelial growth factor (VEGF) functions administered systemically completely block retinal neovascularization. In contrast, selective ocular VEGF depletion has achieved an approx. 50% inhibition of retinal neovascular growth. It is unclear whether a more complete inhibition of new blood vessel development can be obtained with an anti-VEGF therapy localized to the eye. Therefore, the objective of the present study was to determine the effect of local anti-VEGF therapy in a different animal model which closely mimics human ROP. METHODS: Rats were exposed to alternating cycles of high and low levels of oxygen for 14 days immediately after birth; thereafter, they were intravitreally injected with an adenoviral vector expressing a secreted form of the VEGF receptor flt-1 (Ad.sflt), which acts by sequestering VEGF. Contralateral eyes were injected with the control vector carrying the reporter gene expressing beta-galactosidase (Ad.betaGal). RESULTS: At the peak of retinal neovascular growth, i.e. post-natal day 21 (P21), we observed up to 97.5% decrease in retinal neovascularization in animals injected with Ad.sflt. At the end of observation (P28), no significant difference in retinal vessel number was detected in both oxygen-injured and normoxic Ad.sflt-treated retinas compared with untreated or Ad.betaGal-treated retinas. CONCLUSION: Adenoviral-mediated sflt-1 gene transfer induces a near-complete inhibition of ischemia-induced retinal neovascularization in rats without affecting pre-existing retinal vessels.     

Hypoosmotic swelling (HOS) as a screening

Semen from 5 Piedmontese bulls was subjected to the hypoosmotic swelling (HOS) test in order to determine if the results could be correlated to the fertilizing capacity in vitro. Semen was routinely prepared for in vitro fertilization (IVF), with aliquots being sampled after thawing, after separation on a Percoll gradient and after capacitation in a medium containing heparin. The aliquots were added to a fructose-sodium citrate hypoosmotic solution (100 mOsm) and incubated at 37 degrees C for 5 min. At least 200 spermatozoa were observed at x 400 and classified according to the presence or the absence of a swollen tail. After capacitation, spermatozoa were used to fertilize in vitro-matured bovine oocytes (1.5 x10(6) cells/mL); IVF was performed in Fert-TALP medium supplemented with 6 mg/mL BSA and 10 microL/mL heparin in a humidified atmosphere with 5% CO2 and 5% oxygen. Presumptive zygotes were cultured in SOF medium supplemented with 8 mg/mL BSA and amino acids. There were no significant differences in the in vitro fertility of the bulls, but a significant difference was found between bulls in the response to the HOS test. The 3 assays were significantly correlated, while no significant correlation was observed between the percentage of swollen spermatozoa and in vitro fertility. The HOS test does not appear to be sufficiently sensitive to discriminate between semen samples of intermediate fertility like those used in this preliminary research.     

Improved secretion of native human insulin-like growth factor 1 from gas1 mutant Saccharomyces cerevisiae cells

We studied the secretion of recombinant human insulin-like growth factor 1 (rhIGF-1) from transformed yeast cells. The hIGF-1 gene was fused to the mating factor alpha prepro- leader sequence under the control of the constitutive ACT1 promoter. We found that the inactivation of the GAS1 gene in the host strain led to a supersecretory phenotype yielding a considerable increase, from 8 to 55 mg/liter, in rhIGF-1 production.     

Final height in children with idiopathic growth hormone deficiency treated with a fixed dose of recombinant growth hormone

There is no consensus regarding the optimal dosing of recombinant human growth hormone (rhGH) for children with growth hormone deficiency (GHD). Our objective was to evaluate the final adult height (FAH) in children with idiopathic GHD treated with a fixed rhGH dose of 0.18 mg/kg/week. We reviewed all charts of patients with idiopathic GHD treated with rhGH since 1985 who reached FAH. Ninety-six patients were treated for an average of 5.4 years. The mean age was 11.9 years, the mean height -2.87 standard deviation score (SDS) and the mean FAH was -1.04 SDS. Females had a lower predicted adult height than males at the initiation of therapy (-2.0 vs. -1.01 SDS; p = 0.0087) but a higher FAH - predicted adult height (1.08 vs. 0.04 SDS; p = 0.0026). In multiple regression analysis, the FAH SDS was positively related to the midparental height SDS, the height SDS at GH initiation and growth velocity during the first year of therapy, and negatively correlated with peak GH and bone age at initiation (r(2) = 0.51; p < 0.005). Treatment of children with idiopathic GHD with a fixed dose of 0.18 mg/kg/week rhGH is sufficient to reach FAH within 2 SDS of the normal population range (84%) with an average FAH within -0.5 SDS of midparental height.     

Helper-dependent adenovirus for the gene therapy of proliferative retinopathies: stable gene transfer, regulated gene expression and therapeutic efficacy

BACKGROUND: Ocular neovascular disorders, such as diabetic retinopathy and age-related macular degeneration, are the principal causes of blindness in developed countries. Current treatments are of limited efficacy, whereas a therapy based on intraocular gene transfer of angiostatic factors represents a promising alternative. For the first time we have explored the potential of helper-dependent adenovirus (HD-Ad), the last generation of Ad vectors, in the therapy of retinal neovascularization. METHODS: We first analyzed efficiency and stability of intraretinal gene transfer following intravitreous injection in mice. A HD-Ad vector expressing green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter (HD-Ad/GFP) was compared with a first-generation (E1/E3-deleted) Ad vector carrying an identical GFP expression cassette (FG-Ad/GFP). We also constructed HD-Ad vectors expressing a soluble form of the VEGF receptor (sFlt-1) in a constitutive (HD-Ad/sFlt-1) or doxycycline (dox)-inducible (HD-Ad/S-M2/sFlt-1) manner and tested their therapeutic efficacy upon intravitreous delivery in a rat model of oxygen-induced retinopathy (OIR). RESULTS: HD-Ad/GFP promoted long-lasting (up to 1 year) transgene expression in retinal Müller cells, in marked contrast with the short-term expression observed with FG-Ad/GFP. Intravitreous injection of HD-Ad vectors expressing sFlt-1 resulted in detectable levels of sFlt-1 and inhibited retinal neovascularization by more than 60% in a rat model of OIR. Notably, the therapeutic efficacy of the inducible vector HD-Ad/S-M2/sFlt-1 was strictly dox-dependent. CONCLUSIONS: HD-Ad vectors enable stable gene transfer and regulated expression of angiostatic factors following intravitreous injection and thus are attractive vehicles for the gene therapy of neovascular diseases of the retina.     

Pim-1 regulates cardiomyocyte survival downstream of Akt

The serine-threonine kinases Pim-1 and Akt regulate cellular proliferation and survival. Although Akt is known to be a crucial signaling protein in the myocardium, the role of Pim-1 has been overlooked. Pim-1 expression in the myocardium of mice decreased during postnatal development, re-emerged after acute pathological injury in mice and was increased in failing hearts of both mice and humans. Cardioprotective stimuli associated with Akt activation induced Pim-1 expression, but compensatory increases in Akt abundance and phosphorylation after pathological injury by infarction or pressure overload did not protect the myocardium in Pim-1-deficient mice. Transgenic expression of Pim-1 in the myocardium protected mice from infarction injury, and Pim-1 expression inhibited cardiomyocyte apoptosis with concomitant increases in Bcl-2 and Bcl-X(L) protein levels, as well as in Bad phosphorylation levels. Relative to nontransgenic controls, calcium dynamics were significantly enhanced in Pim-1-overexpressing transgenic hearts, associated with increased expression of SERCA2a, and were depressed in Pim-1-deficient hearts. Collectively, these data suggest that Pim-1 is a crucial facet of cardioprotection downstream of Akt.     

Infection of bovine oviduct cell cultures with Chlamydophila abortus

Bovine infertility is a major cause of loss in the livestock industry. In the present study bovine oviduct cell cultures were infected with a Chlamydophila abortus strain. A direct evaluation of infection was performed by means of May Grünwald-Giemsa and immunocytochemistry for chlamydial LPS, which revealed inclusion bodies and vacuolisation. SEM and TEM analysis of infected cells showed various degrees of cell damage and conglutination of microvilli. This finding suggests that cattle infertility may result from an alteration of oviduct environment caused by multiplication of C. abortus. This microorganism, among other infectious agents, could be considered a potential causative agent of bovine infertility.     

The effect of Mycoplasma mycoides ssp. mycoides LC of bovine origin on in vitro fertilizing ability of bull spermatozoa and embryo development

Several Mycoplasma species may adversely affect bovine spermatozoa viability and embryo development. Mycoplasma mycoides ssp. mycoides large-colony (LC) has been isolated from naturally aborted bovine fetuses and from bull semen. The objective of this study was to evaluate whether M. mycoides ssp. mycoides LC contaminated bovine ejaculates could (i) impair in vitro fertilizing ability of bull spermatozoa, (ii) impair embryo development, and (iii) evaluate potential spread by reproductive technologies. In the present study, spermatozoa of 10 fertile bulls were contaminated with M. mycoides ssp. mycoides LC, at a final concentration of 1.5 million CFU/ml and incubated for 60 min before evaluating spermatozoa motility and acrosome reaction inducibility with calcium ionophore. In addition, in vitro contaminated semen of a bull previously shown to have a good in vitro fertilizing ability, was used in an IVF procedure. Embryo development stage on Day-7 of culture was evaluated. Spermatozoa and embryos at morula and blastocyst stages were routinely processed for transmission electron microscopy observation. Both mean total and progressive motility decreased (P < 0.01 ) upon spermatozoa incubation with Mycoplasma. One-hour incubation with calcium ionophore increased the percentage of acrosome-reacted spermatozoa, although Mycoplasma contamination reduced calcium ionophore treatment efficacy (P < 0.05). Ultrastructurally, Mycoplasma microorganisms appeared as moderately electron-dense sphere-shaped particles, adhering to cell membranes. Sperm mid-piece sections showed numeric aberrations of the central singlets such as nine + zero or nine + one of the axonemal complex. Further morphological abnormalities included partial or total absence of dinein arms and radial fibers, with lack of the bridge and the central ring in 35.00 +/- 4.20% of contaminated cells, whereas these abnormalities were not observed in uninfected ones. The IVF trials showed that two-four cell blocks were higher (P < 0.05) in the infected group. Ultrastructure of Day-7 contaminated embryos showed Mycoplasma particles adhering and infiltrating the outer layer of the zona pellucida. Our investigations suggest that M. mycoides ssp. mycoides LC contaminating the bovine ejaculate induced adverse effects on in vitro spermatozoa-fertilizing ability and embryonic development. Some satisfactory quality transferable embryos could be produced in contaminated IVF systems. This could imply a potential transmission of this microorganism through reproductive technologies.     

Evaluation of dog semen quality after slow (biological freezer) or rapid (nitrogen vapours) freezing

Three ejaculates were collected from each of five dogs. After initial evaluation, the sperm-rich fractions were diluted to 100 x 10(6) spermatozoa x mL(-1) in two steps with an egg yolk-TRIS extender containing a final concentration of 5% glycerol and 0.5% Equex STM paste. Half of the 0.5 mL straws obtained from each ejaculate were frozen on nitrogen vapours (4 cm above the liquid surface) ("rapid freezing"), while the other half was frozen in a biological freezer at a rate of 0.5 degrees C x min(-1) between 5 degrees C and -10 degrees C and of 8 degrees C x min(-1) between -10 degrees C and -60 degrees C, followed by immersion in liquid nitrogen ("slow freezing"). After an average storage of 30 days, the straws were thawed in a water-bath at 37 degrees C for 1 min. Progressive motility was subjectively estimated hourly for 8 h on semen incubated at 38 degrees C. Immediately after thawing and after 2 h of incubation, motility parameters were also measured by a motility analyser. Sperm membrane function and chromatin stability were assessed immediately post-thaw, using the hypo-osmotic swelling test and acridine orange staining, respectively. Slow freezing significantly improved total post-thaw motility, which showed a slower decline over time, although spermatozoal average path and straight line velocity were lower compared to the fast rate. Also the number of intact membrane spermatozoa was significantly higher in slow-frozen samples while the proportion of spermatozoa with single-stranded DNA was minimal after both freezing procedures.