Effect of pentoxifylline treatment on testicular perfusion and semen quality in Miniature horse stallions

The objective was to investigate the effects of pentoxifylline (PTX) on testicular perfusion and sperm production in stallions. In a preliminary study, six mature Miniature horse stallions were given 0, 8.5, or 17.0 mg/kg of PTX orally, twice daily, for 3 d. Total Arterial Blood Flow Rate (TABFR) was higher (P < 0.05) in all treated versus control stallions during and after treatment. Two months later (during the fall and winter), the same stallions received either 0 or 17 mg/kg of PTX orally, twice daily for 60 d. Resistance and pulsatility indices (RI and PI, respectively) decreased in PTX-treated stallions between Treatment 1 and Post-treatment periods. Arterial diameter, as well as Total Arterial Blood Flow (TABF), decreased in controls between Baseline and Treatment 1 (P < 0.05). A similar decrease in arterial diameter was delayed in Group TREATED, but reached significance during Post-treatment (P < 0.05), whereas TABF did not change in this group. Furthermore, TABFR had a transient tendency to increase during Treatment 1 (P < 0.1), whereas it steadily decreased in controls and reached significance in the Post-treatment period (P < 0.05). Both RI and PI were negatively correlated with end diastolic velocity (EDV) in both groups (P < 0.0001). There were positive correlations between RI and peak systolic velocity (PSV) in treated stallions during Treatment 1 (RI: r = 0.53, P = 0.021; PI: r = 0.59, P = 0.007). Also, there were negative correlations between Time Averaged Maximum Velocity (TAMAX) and Doppler indexes in treated stallions during Treatment 2 period (RI: r = −0.49, P = 0.006; PI: r = −0.47, P = 0.008), and during Post-treatment periods (RI: r = −0.40, P = 0.049; PI: r = −042, P = 0.039). Transient hydrocele occurred in all treated stallions (a potential complication of high-dose PTX). Semen end points were not significantly affected by PTX treatment. In conclusion, PTX delayed the seasonal decrease of testicular perfusion in stallions. Sperm quality and quantity were not significantly affected; perhaps they would have been enhanced by prolonged treatment.     

Equilin and equilenin biosynthesis. Stereochemistry of aromatization of 3-hydroxy-3,5,7-androstatrien-17-one by horse placenta

The metabolic pathway leading to equilin and equilenin biosynthesis in the pregnant mare is different from that of estrone and estradiol and it is apparently cholesterol-independent. The precise precursors and intermediates and the stereomechanism of equine placental aromatization have not been established. [1,2-3H, 4-14C]3-Hydroxy-3,5,7-androstatrien-17-one was synthesized as a potential substrate and the 3H-distribution was analyzed by biochemical and chemical derivatization methods. The substrate was converted to equilin, equilenin and Heard's ketone by horse placental microsomes with a sp. act. of 74, 18 and 2.8 pmol/h/mg, respectively, and only to equilin by human placental microsomes with a rate of 26 pmol/h/mg. Analysis of the loss of 3H-labeling during aromatization showed the stereospecific 17 beta,2 beta-cis hydrogen elimination for equine estrogen biosynthesis both by horse and human placental microsomes. This is the same as for estrone and estradiol biosynthesis by both placentas. The biosynthesis of Heard's ketone, a non-phenolic ring-B aromatic C18 steroid, by horse placental microsomes was found to involve none of the four hydrogens at C-1 and C-2. This refutes the previous postulate that Heard's ketone arises from equilenin by reduction of the ring-A.     

The compounds from the hollyhock extract (Althaea rosea Cav. var. nigra) affect the aromatization in rat testicular cells in vivo and in vitro

Among medicinal plants, extract from the hollyhock flowers is a source of antocyanides and flavonoids. The latter compounds belong, among others, to phytoestrogens (plant-derived dietary estrogens). The important role of estrogens in the testis is now well documented, and phytoestrogens, which may act as estrogen agonists or estrogen antagonists can also alter the reproductive function of the male. The aim of this study was to show whether the exposure of male rats to the aqueous hollyhock extract could affect the process of aromatization in their testes and in cultured Leydig cells. This was investigated by immunocytochemistry and radioimmunological assays. Immunoreactivities for aromatase and estrogen receptor beta were weaker both in testicular sections and cultured Leydig cells after hollyhock extract administration when compared to the controls, while the intensity of immunoreaction for estrogen receptor alpha remained unchanged. A lower level of estradiol secreted by cultured Leydig cells from the experimental group positively correlated with a direct inhibition of aromatase activity. Additionally, a quantitative analysis of flavonoid fraction from the hollyhock extract revealed the presence of quercetin and kaempferol. It seems that a weak antiestrogenic activity of flavonoid compounds present in the hollyhock extract is mediated through aromatase and estrogen receptor beta rather than by estrogen receptor alpha.     

The site of aromatization in the mouse cryptorchid testis

Using the mouse cryptorchid model, degenerations of germ cells were observed as well as a reduced size of seminiferous tubules, while the area of the interstitial tissue increased. Aromatase, the enzyme responsible for the conversion of androgens into oestrogens, was immunolocalized in Leydig cells and in germ cells from both scrotal and abdominal testes, and in Sertoli cells only in a control testis. In the cryptorchid testis, aromatase was strongly expressed in a few tubules, including those spermatids that were still present. Other cells inside the tubules were negative for aromatase. In both testes, oestrogen receptors alpha were expressed only in Leydig cells. Strong aromatase expression in germ cells indicates an additional source of oestrogens in the testis besides the interstitial tissue.     

Down-regulation of the interferon receptor

The binding of 125I-labeled alpha A interferon to human lymphoblastoid Daudi cells decreased when these cells were incubated with unlabeled alpha or beta interferon. This decrease could not be accounted for by the occupancy of interferon receptors with unlabeled interferon and it apparently resulted from the loss or down-regulation of receptors. The binding activity gradually increased when Daudi cells were incubated in fresh medium after a treatment with interferon, but inhibition of protein synthesis with cycloheximide prevented this recovery. Treatment of Daudi cells with this inhibitor resulted in the loss of half the interferon binding activity within 5 h. These findings suggested that the interferon receptors turn over at a basal rate in interferon-free medium and at an increased rate in cells incubated with interferon. The dose-response for the down-regulation was investigated by treating Daudi cells with different concentrations of alpha interferon. Down-regulation was observed in cells treated with relatively low doses of interferon, sufficient to elicit a biological response. The synthesis of the enzyme (2',5')oligo(A) polymerase was induced at the lowest interferon concentrations tested which caused receptor down-regulation.     

Sweating in the intact horse and isolated perfused horse skin

1. In intact horses, heat-induced sweating occurred initially as pulses, then as a continuous, synchronously fluctuating discharge. 2. I.V. adrenaline (Adr) induced sweating immediately; isoprenaline (Isop) elicited sweating after a delay; and phenylephrine (PhE) had no sudorific effect. 3. In isolated perfused skin, PhE induced an immediate small sweat discharge, Isop a slower sustained output and Adr a biphasic discharge. alpha- and beta-adrenergic antagonists blocked the first and second phases, respectively, of Adr-induced sweating. 4. The observed sweating patterns are consistent with independent activation of alpha-adrenergic myoepithelium and beta-adrenergic secretory cells in the sweat glands. 5. Microcirculatory changes apparently also influenced sweat discharge.     

Synthesis and aromatization of 19-norandrogens in the stallion testis

The results of the measurement of 19-nortestosterone in the testiscular artery and vein of the stallion, the very low levels of this steroid in the peripheral blood of geldings and the similar patterns of increase in the peripheral levels of 19-nortestosterone and testosterone after hCG stimulation, show that 19-nortestosterone, like testosterone, is essentially synthesized in the testis. This testicular origin was confirmed by the ability of testicular tissue to synthesize 19-norandrogens from [4-14C]androgens in vitro. 19-Nortestosterone was 50% conjugated in the peripheral blood and almost entirely conjugated after biosynthesis in vitro. The sequence of appearance of steroids in the peripheral blood after a single injection of 10,000 IU hCG suggests that, in the equine testis, 19-norandrogens are produced by a specific C10-19 desmolase (estrene synthetase), stimulable by hCG. 19-Nortestosterone was aromatized into estradiol-17 beta by stallion testicular microsomes. The affinity of the aromatase for 19-nortestosterone was very low compared to that for testosterone. At low and presumably physiological levels, and at a high testosterone/19-nortestosterone ratio, testosterone did not inhibit 19-nortestosterone aromatization by more than 53%. Thus, 19-nortestosterone may be aromatized in vivo in the testis in spite of the endogenous concentrations of androgens. However, the low velocity of 19-nortestosterone aromatization by testicular microsomes at roughly physiological concentrations suggests that 19-norandrogen aromatization may only participate slightly in the testicular estrogen production. These results suggest that in the equine testis, two aromatizing enzyme systems may exist: one which aromatizes both androgens and 19-norandrogens, and a minority system more specific for 19-norandrogens.     

B-ring aromatization of estrogen derivatives

A novel method of B-ring aromatization of steroid derivatives is reported. Addition of BrCl in methanol to a non-aromatic B-ring double bond results in a rapid double elimination generating the aromatic B-ring. This procedure represents an effective method for conversion of an equilin to an equilenin nucleus.     

Stereochemistry of 1,2-hydrogen loss during aromatization in the brain

The stereochemistry of hydrogen loss from C-1 and C-2 during aromatization in rat brain was studied using androstenedione containing a known distribution of isotopic label. Comparison of the tritium content of the estrone obtained from the aromatization of androstenedione labeled predominantly in the 1 alpha,2 alpha positions with that in estrone obtained from a parallel incubation using substrate with label in the 1 beta,2 beta orientation gave an estrone alpha/beta ratio of 3.6. This ratio compares with a calculated value of 4.3 for an aromatization mechanism involving loss of the 1 beta,2 beta-hydrogens. The distortion from the predicted value is due to the loss of tritium from the alpha-substrate which is unrelated to aromatization. The ratio determined experimentally is compatible with 2 beta-tritium loss since random or alpha-elimination from C-2 would yield alpha/beta ratios of 2.2 and 1.3 respectively. In an analogous manner the stereochemistry of tritium loss at C-1 was determined using [1 alpha-3H] and [1 beta-3H]androstenedione. The alpha/beta ratio of the isolated estrone was 3.6 which is in good agreement with the calculated value of 3.3 for 1 beta-tritium elimination. Our results therefore show that estrogen formation in the brain occurs with the same stereospecificity of hydrogen loss at C-1 and C-2 as in placental microsomes.     

Electroacupuncture enhances extragonadal aromatization in ovariectomized rats

Background  

Repeated electroacupuncture (EA) stimulation is known to stimulate the activity of the hypothalamus-pituitary-adrenal axis, and to enhance the circulation level of estrogen in the ovariectomized rats. To explore the source of the increased circulation estrogen, the extragonadal aromatization was detected.     

Inhibition of androgen aromatization in human breast cancer

The inhibitory activity of aminoglutethimide, 4-hydroxy-androstenedione and 7α-(4'-amino) phenylthioandrostenedione on human placental and mammary tumor aromatase activity was examined. [¹⁴C]-Androstenedione was incubated with placental microsomes and mammary tumor homogenates in the presence of an NADPH generating system with or without inhibitor. All three inhibitors were equally effective in inhibiting microsomal placental aromatase at various inhibitor concentrations. Inhibitions of mammary tumor aromatase using 12 μM inhibitor concentrations were essentially similar to results on placental aromatase inhibition and ranged from 81–97% inhibition. These findings are discussed in regard to the potential clinical use of aromatase inhibitors in the treatment of estrogen dependent metastatic breast carcinoma.     

Inbreeding in the Thoroughbred horse

Changes in the inbreeding coefficient, F, in the Thoroughbred horse over the past 45 years have been investigated by genotyping 467 Thoroughbred horses (born between 1961 and 2006) using the Illumina Equine SNP50 bead chip, which comprises 54,602 SNPs uniformly distributed across the equine genome. The Spearman rank correlation coefficient, r, between the year of birth and F was estimated. The results indicate that inbreeding in Thoroughbreds has increased over the past 40 years, with r = 0.24, P < 0.001 demonstrating that there is a highly significant, though relatively weak correlation between the year of birth and inbreeding coefficients. Interestingly, the majority of the increase in inbreeding is post-1996 and coincides with the introduction of stallions covering larger numbers of mares.     

Exercise-induced hypercapnia in the horse

The effects of exercise intensity and duration on blood gases in thoroughbred horses were studied to characterize the apparent exercise-induced failure in pulmonary gas exchange that occurs in these animals. In response to 2 min of exercise, arterial CO2 tension (PaCO2) decreased in mild and moderate exercise, returned to normocapnic levels in moderate to heavy exercise, and rose 5-10 Torr above resting values during very heavy exercise when CO2 production (VCO2) exceeded 20 times the resting value, and mixed venous CO2 tension approximated 140 Torr. Exercise-induced hypoxemia occurred at the onset of heavy exercise and was associated with the absence of a hyperventilatory response and an alveolar-arterial PO2 difference that increased four to six times above rest with very heavy exercise. PaCO2 was related to VCO2 but not fb, as changes in breathing frequency (fb) of 8-20 breaths/min at comparable VCO2 did not affect PaCO2. Prolonging very heavy exercise from 2 to 4 min caused a severe metabolic acidosis (arterial pH less than 7.15) and hypoxemia was maintained; however, CO2 was no longer retained, as PaCO2 gradually fell to below resting levels, due to an increased tidal volume at constant fb. We conclude that a truly compensatory hyperventilation to very heavy exercise in the horse is not achieved because of the excessive volumes and flow rates required by their extraordinarily high VCO2 and VO2. On the other hand, the frank CO2 retention during short-term high-intensity exercise occurs even though the horse is not apparently mechanically obligated to tolerate it.     

Embryo technologies in the horse

Recent studies demonstrated that zwitterionic buffers could be used for satisfactory storage of equine embryos at 5 degrees C. The success of freezing embryos is dependent upon size and stage of development. Morulae and blastocysts <300 microm can be slowly cooled or vitrified with acceptable pregnancy rates after transfer. The majority of equine embryos are collected from single ovulating mares, as there is no commercially available product for superovulation in equine. However, pituitary extract, rich in FSH, can be used to increase embryo recovery three- to four-fold. Similar to human medicine, assisted reproductive techniques have been developed for the older, subfertile mare. Transfer of in vivo-matured oocytes from young, healthy mares into a recipient's oviduct results in a 70-80% pregnancy rate compared with a 30-40% pregnancy rate when the oocytes are from older, subfertile mares. This procedure can also be used to evaluate in vitro maturation systems. In vitro production of embryos is still quite difficult in the horse. However, intracytoplasmic sperm injection (ICSI) has been used to produce several foals. Cleavage rates of 60% and blastocyst rates of 30% have been reported after ICSI of in vitro-matured oocytes. Gamete intrafallopian tube transfer (GIFT) is a possible treatment for subfertile stallions. Transfer of in vivo-matured oocytes with 200,000 sperm into the oviduct of normal mares resulted in a pregnancy rate of 55-82%. Oocyte freezing is a technique that has proven difficult in most species. However, equine oocytes vitrified in a solution of ethylene glycol, DMSO, and Ficoll and loaded onto a cryoloop resulted in three pregnancies of 26 transfers and two live foals produced. Production of a cloned horse appears to be likely, as several cloned pregnancies have recently been produced.