In utero betamethasone affects 3ß-hydroxysteroid dehydrogenase and inhibin-α immunoexpression during testis development.

Prenatal glucocorticoids, commonly used in women at risk of preterm delivery, can predispose the newborn to disease in later life. Since male reproductive function is likely to reflect testis development during fetal life, we studied the effects of prenatal glucocorticoids on two key intra-testicular factors that play roles in cellular proliferation and differentiation, 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and inhibin-α. Pregnant sheep (n=42) were treated with betamethasone (0.5 mg/kg) or saline (control) at 104, 111 and 118 days of gestation (DG). Testicular tissue was sampled from fetuses at 121 and 132DG, and from lambs at 45 and 90 postnatal days (PD). Within the betamethasone treated group, 3ß-HSD immunostaining area was greater at 121DG than at 90PD (P=0.04), but the intensity of immunostaining was higher at 90PD than at 121DG (P=0.04), 132DG (P=0.04) and 45PD (P=0.03). Control animals showed no changes in 3ß-HSD area or intensity of immunostaining. No significant differences were observed between treated and control animals in immunostaining area, but immunostaining was more intense in the treated group than in the control group at 90PD (P=0.03). For inhibin-α, the proportion of immunostaining area declined in treated offspring from 121DG to 45PD, in contrast to control values, but recovered fully by 90PD, concomitantly with the onset of spermatogenesis. In conclusion, prenatal betamethasone increased the postnatal testicular expression of inhibin-α but reduced the expression of 3ß-HSD. These effects could compromise androgen-mediated testicular development and therefore adult capacity for spermatogenesis.

Accessibility of blood affects the attractiveness of cattle to horn flies.

The burden of infestation of the horn fly, Haematobia irritans (Linnaeus) (Diptera: Muscidae), differs among bovines within the same herd. We hypothesized that these differences might be related to the epidermal thickness of the cattle and the blood intake capacity of the fly. Results showed that dark animals carried more flies and had a thinner epidermis than light-coloured animals, which was consistent with the greater haemoglobin content found in flies caught on darker cattle. Similarly, epidermal thickness increased with body weight, whereas haemoglobin content decreased. Overall, we suggest that accessibility of blood is a factor that partially explains cattle attractiveness to flies.

Apoptosis-related protein expression during pre- and post-natal testicular development after administration of glucocorticoid in utero in the sheep.

Pre-natal glucocorticoids are used in women at risk of preterm delivery to induce foetal lung maturation. However, glucocorticoids can produce negative outcomes for other tissues such as the reproductive system. We therefore tested the effects of pre-natal betamethasone on testicular morphology and apoptotic protein immune expression during pre- and post-natal development. Pregnant ewes (n = 42) bearing singleton male foetuses were randomly allocated to receive intramuscular injections of saline or betamethasone (0. 5 mg/kg) at 104, 111 and 118 days of gestation (DG). Testes were collected at 121 and 132 DG, and at 45 and 90 post-natal days (PD) and subjected to morphometric analysis (volume densities of sex cords and interstitial tissues; sex cord diameter). Immunohistochemistry (% stained area) was used to assess active caspase-3, Bax, Bcl-2 and cell-cycle proteins (PCNA). Compared with control values, betamethasone treatment decreased sex cord diameter at 121 DG, 45 and 90 PD, and sex cord volume at 90 PD. Active caspase-3 was decreased by betamethasone at 121 DG and 90 PD, but Bax was increased in all betamethasone groups. Bcl-2 and PCNA decreased in the betamethasone groups at 121 DG and 45 PD, but increased at 132 DG and 90 PD. We conclude that high levels of pre-natally administered glucocorticoid reduce foetal testicular development, perhaps via changes in the balance between pro- and anti-apoptotic proteins and cell-cycle proteins. These outcomes could compromise the future spermatogenic potential of male offspring.

Effects of pre-natal glucocorticoids on testicular development in sheep.

We tested the hypothesis that acute pre-natal exposure to high levels of synthetic glucocorticoid (betamethasone) would alter fetal testicular development through actions on gonadal glucocorticoid receptors (GRs). Pregnant Merino ewes bearing singleton male fetuses (n = 24) were allocated randomly among four equal groups to be injected intramuscularly with saline or betamethasone (0.5 mg/kg) either on day 109 of gestation or on both day 109 and day 116 of gestation. Fetal testes were collected at post-mortem, 5 days after each treatment. The volume of interstitial tissue and the volume, length and diameter of the sex cords were measured, and Sertoli cells and gonocytes were counted. For cord volume and interstitial tissue volume, control testes demonstrated maturational changes as fetal age advanced from 109 to 116 days of gestation. For that period, the single injection of betamethasone significantly reduced Leydig cell proliferation (P < 0.05), but had no effect on Sertoli cell numbers. Immunohistochemistry was used to localize GR and proliferating cell nuclear antigen in testicular cells. GR immunoexpression in Leydig cells was higher in fetuses exposed to betamethasone at 109 days of gestation than in control fetuses. Sertoli cells showed low levels of GR. It was concluded that, during mid-gestation, a brief period of glucocorticoid treatment could affect testicular development in male sheep fetuses. The mechanism probably involves direct effects on Leydig cells, as these cells express extra-GR in response to the treatment. Sertoli cells seem to produce less GR than Leydig cells, perhaps explaining their lack of response to betamethasone. These outcomes may have important implications for future fertility in male offspring.

Nutritional management during fetal and postnatal life, and the influence on testicular stereology and Sertoli cell numbers in Corriedale ram lambs.

The aim of the experiment was to determine whether supplementation of the lamb-ewe unit during intra-uterine and postnatal life affects testicular stereology, particularly Sertoli cell numbers, in 120 pregnant Corriedale ewes grazed either native pastures (control group) or improved pastures+grain supplement (treated group). Ewes bearing single ram lambs were maintained under the same feeding regime until lambs were castrated (99 days of age). Body weight, testosterone and FSH blood serum levels were recorded at 45, 75 and 99 days of age. Body weight was higher (P<0.01) in the treated group from birth on. Serum testosterone values did not differ between groups. Serum FSH values tended to differ at 45 days of age (P<0.06). Testicular weight and testes histology showed earlier pubertal development and a tendency for higher Sertoli cell numbers in the treated (supplemented) group. This tendency may indicate that extensively reared lambs supplemented during fetal and postnatal life have higher testicular growth and sperm production in later life.

Influence of pre- and post-pubertal grazing regimes on adult testicular morphology in extensively reared corriedale rams.

The aim of the present study was to determine whether pre- and post-pubertal young rams on different grazing regimes, resulting in differences in live weight (LW), would show corresponding differences in testicular growth or testicular morphometry that could influence the reproductive traits of these rams upon reaching adulthood. Forty-one spring-born Corriedale rams were reared on either native pasture (low feeding level, Group L, n=22) or improved pasture (higher feeding level, Group H, n=19) from 1 to 7 months of age. Thereafter, half the animals in the native-pasture group were placed on improved pasture and vice versa, thus creating an additional four differential-grazing treatment groups (Groups LL, n=11; LH, n=11; HL, n=10; and HH, n=9). Animals were managed in this way until 18 months of age. Half the animals from each group were then castrated and their testes were subjected to morphometric analysis. The remaining animals (Groups LL, n=6; LH, n=6; HL, n=5; and HH, n=4) were managed together until 30 months of age (from 18 to 27 months on native pastures and from 27 to 30 months of age on improved pastures, at a stocking rate of two to three rams per hectare), whereupon they were also castrated for testicular morphometry. LW and scrotal circumference (SC) were recorded every 60 days. The stereological analysis of testicular parenchyma included counts of elongated spermatids and Sertoli cells. Differences (P<0.001) in LW were observed between feeding levels, even at 30 months of age. Differences (P<0.001) in SC existing at the end of the differential treatment (18 months of age) disappeared (n.s.) soon after. Most differences (P<0.05) in testicular morphometry existing at the end of the differential treatments were no longer significant 1 year later. It is concluded that changes in grazing management during pre- and post-pubertal periods can induce short-lived differences in testicular post-natal growth in Corriedale rams but do not influence testicular morphology or function later in life.

An immunohistochemical study on the regulation of estrogen receptor alpha by estradiol in the endometrium of the immature ewe.

The effects of estradiol-17beta (E2) on the expression of estrogen receptor alpha (ERalpha) in stromal and epithelial cells of endometrium in prepubertal lambs were investigated. Twenty three-month-old lambs were treated or not treated with one, two or three i.m. injections of E2 (1 microg x kg(-1)) in corn oil at intervals of 24 h. Lambs were slaughtered 12 or 24 h after the last injection. An immunohistochemical technique was used to visualize ERalpha immunostaining which was then analyzed quantitatively by a computer imaging analysis system. Seven endometrial compartments defined by cell type and location were analyzed separately. Positive staining of ERalpha was seen in the nuclei of stromal and epithelial cells. Glandular epithelium located next to the myometrium was stained more intensely than that next to the luminal epithelium and this phenomenon was maintained during treatment. Significantly less immunostaining was found in stromal cells 12 and 24 h after the first injection compared to the control group. A similar pattern was found in the glandular epithelium, although the decrease was more pronounced and the restoration of ERalpha was faster. This study shows that E2 treatment down regulates ERalpha in the endometrium temporarily in both stromal and epithelial cells, but the characteristics of this effect seems to be cell type specific.

Influence of grazing management on the seasonal change in testicular morphology in Corriedale rams.

The present study was conducted: (a) to determine the degree of seasonal variation in testis stereology in Corriedale rams between autumn and winter; (b) to test the hypothesis that testis stereology of Corriedale rams grazing native pastures during autumn and winter would differ from those of Corriedale rams grazing sown pastures and supplemented with grain during the same period; and (c) to determine whether Sertoli cell numbers differ in adult rams between the breeding season (autumn) and the following non-breeding season (winter). Twenty experimental animals were studied. Six rams (autumn control group, C-A) that had been grazing on native pasture (stocking rate = 2-3 animals ha(-1)) were castrated at the beginning of the experiment (March, early autumn). Seven rams (winter control group, C-W) continued to graze on native pasture at the same stocking rate until the end of the experiment (August, late winter). Another seven rams (treated group, T) grazed on improved pasture (stocking rate = 1-2 animals ha(-1)) and were supplemented with 1 kg grain ram(-1) day(-1) until the end of the experiment. Live weight, scrotal circumference, serum testosterone concentration and selected testicular stereological parameters were measured. The treatment did not impede the winter reduction in testicular activity and reduced its magnitude slightly (group T) compared with controls (group C-W). Sertoli cell numbers were higher in autumn (group C-A) than in winter, both on native (group C-W) and sown pastures (group T). Diminishing Sertoli cell numbers between autumn and the following winter suggest the occurrence of that Sertoli cell death during this period. The results indicate that, although the reproductive activity of Corriedale rams is moderately seasonal, a restricted change in grazing and grain supplementation can only modify it to a limited extent.