Prenatal cortisol exposure impairs adrenal function but not glucose metabolism in adult sheep.

Adverse environmental conditions before birth are known to programme adult metabolic and endocrine phenotypes in several species. However, whether increments in fetal cortisol concentrations of the magnitude commonly seen in these conditions can cause developmental programming remains unknown. Thus, this study investigated the outcome of physiological increases in fetal cortisol concentrations on glucose-insulin dynamics and pituitary-adrenal function in adult sheep. Compared with saline treatment, intravenous fetal cortisol infusion for 5 days in late gestation did not affect birthweight but increased lamb body weight at 1-2 weeks after birth. Adult glucose dynamics, insulin sensitivity and insulin secretion were unaffected by prenatal cortisol overexposure, assessed by glucose tolerance tests, hyperinsulinaemic-euglycaemic clamps and acute insulin administration. In contrast, prenatal cortisol infusion induced adrenal hypo-responsiveness in adulthood with significantly reduced cortisol responses to insulin-induced hypoglycaemia and exogenous adrenocorticotropic hormone (ACTH) administration relative to saline treatment. The area of adrenal cortex expressed as a percentage of the total cross-sectional area of the adult adrenal gland was also lower after prenatal cortisol than saline infusion. In adulthood, basal circulating ACTH but not cortisol concentrations were significantly higher in the cortisol than saline-treated group. The results show that cortisol overexposure before birth programmes pituitary-adrenal development with consequences for adult stress responses. Physiological variations in cortisol concentrations before birth may, therefore, have an important role in determining adult phenotypical diversity and adaptability to environmental challenges.

Glucocorticoid maturation of mitochondrial respiratory capacity in skeletal muscle before birth.

In adults, glucocorticoids act to match the supply and demand for energy during physiological challenges, partly through actions on tissue mitochondrial oxidative phosphorylation (OXPHOS) capacity. However, little is known about the role of the natural prepartum rise in fetal glucocorticoid concentrations in preparing tissues for the increased postnatal energy demands. This study examined the effect of manipulating cortisol concentrations in fetal sheep during late gestation on mitochondrial OXPHOS capacity of two skeletal muscles with different postnatal locomotive functions. Mitochondrial content, biogenesis markers, respiratory rates and expression of proteins and genes involved in the electron transfer system (ETS) and OXPHOS efficiency were measured in the biceps femoris (BF) and superficial digital flexor (SDF) of fetuses either infused with cortisol before the prepartum rise or adrenalectomised to prevent this increment. Cortisol infusion increased mitochondrial content, biogenesis markers, substrate-specific respiration rates and abundance of ETS complex I and adenine nucleotide translocator (ANT1) in a muscle-specific manner that was more pronounced in the SDF than BF. Adrenalectomy reduced mitochondrial content and expression of PGC1α and ANT1 in both muscles, and ETS complex IV abundance in the SDF near term. Uncoupling protein gene expression was unaffected by cortisol manipulations in both muscles. Gene expression of the myosin heavy chain isoform, MHCIIx, was increased by cortisol infusion and reduced by adrenalectomy in the BF alone. These findings show that cortisol has a muscle-specific role in prepartum maturation of mitochondrial OXPHOS capacity with important implications for the health of neonates born pre-term or after intrauterine glucocorticoid overexposure.

Development and thyroid hormone dependence of skeletal muscle mitochondrial function towards birth.

KEY POINTS: Skeletal muscle energy requirements increase at birth but little is known regarding the development of mitochondria that provide most of the cellular energy as ATP. Thyroid hormones are known regulators of adult metabolism and are important in driving several aspects of fetal development, including muscle fibre differentiation. Mitochondrial density and the abundance of mitochondrial membrane proteins in skeletal muscle increased during late gestation. However, mitochondrial functional capacity, measured as oxygen consumption rate, increased primarily after birth. Fetal hypothyroidism resulted in significant reductions in mitochondrial function and density in skeletal muscle before birth. Mitochondrial function matures towards birth and is dependent on the presence of thyroid hormones, with potential implications for the health of pre-term and hypothyroid infants. ABSTRACT: Birth is a significant metabolic challenge with exposure to a pro-oxidant environment and the increased energy demands for neonatal survival. This study investigated the development of mitochondrial density and activity in ovine biceps femoris skeletal muscle during the perinatal period and examined the role of thyroid hormones in these processes. Muscle capacity for oxidative phosphorylation increased primarily after birth but was accompanied by prepartum increases in mitochondrial density and the abundance of electron transfer system (ETS) complexes I-IV and ATP-synthase as well as by neonatal upregulation of uncoupling proteins. This temporal disparity between prepartum maturation and neonatal upregulation of mitochondrial oxidative capacity may protect against oxidative stress associated with birth while ensuring energy availability to the neonate. Fetal thyroid hormone deficiency reduced oxidative phosphorylation and prevented the prepartum upregulation of mitochondrial density and ETS proteins in fetal skeletal muscle. Overall, the data show that mitochondrial function matures over the perinatal period and is dependent on thyroid hormones, with potential consequences for neonatal viability and adult metabolic health.

A physiological increase in maternal cortisol alters uteroplacental metabolism in the pregnant ewe.

KEY POINTS: Fetal nutrient supply is dependent, in part, upon the transport capacity and metabolism of the placenta. The stress hormone, cortisol, alters metabolism in the adult and fetus but it is not known whether cortisol in the pregnant mother affects metabolism of the placenta. In this study, when cortisol concentrations were raised in pregnant sheep by infusion, proportionately more of the glucose taken up by the uterus was consumed by the uteroplacental tissues while less was transferred to the fetus, despite an increased placental glucose transport capacity. Concomitantly, the uteroplacental tissues produced lactate at a greater rate. The results show that maternal cortisol concentrations regulate uteroplacental glycolytic metabolism, producing lactate for use in utero. Prolonged increases in placental lactate production induced by cortisol overexposure may contribute to the adverse effects of maternal stress on fetal wellbeing. ABSTRACT: Fetal nutrition is determined by maternal availability, placental transport and uteroplacental metabolism of carbohydrates. Cortisol affects maternal and fetal metabolism, but whether maternal cortisol concentrations within the physiological range regulate uteroplacental carbohydrate metabolism remains unknown. This study determined the effect of maternal cortisol infusion (1.2 mg kg-1  day-1 i.v. for 5 days, n = 20) on fetal glucose, lactate and oxygen supplies in pregnant ewes on day ∼130 of pregnancy (term = 145 days). Compared to saline infusion (n = 21), cortisol infusion increased maternal, but not fetal, plasma cortisol (P < 0.05). Cortisol infusion also raised maternal insulin, glucose and lactate concentrations, and blood pH, PCO2 and HCO3- concentration. Although total uterine glucose uptake determined by Fick's principle was unaffected, a greater proportion was consumed by the uteroplacental tissues, so net fetal glucose uptake was 29% lower in cortisol-infused than control ewes (P < 0.05). Concomitantly, uteroplacental lactate production was > 2-fold greater in cortisol- than saline-treated ewes (P < 0.05), although uteroplacental O2 consumption was unaffected by maternal treatment. Materno-fetal clearance of non-metabolizable [3 H]methyl-d-glucose and placental SLC2A8 (glucose transporter 8) gene expression were also greater with cortisol treatment. Fetal plasma glucose, lactate or α-amino nitrogen concentrations were unaffected by treatment although fetal plasma fructose and hepatic lactate dehydrogenase activity were greater in cortisol- than saline-treated ewes (P < 0.05). Fetal plasma insulin levels and body weight were also unaffected by maternal treatment. During stress, cortisol-dependent regulation of uteroplacental glycolysis may allow increased maternal control over fetal nutrition and metabolism. However, when maternal cortisol concentrations are raised chronically, prolonged elevation of uteroplacental lactate production may compromise fetal wellbeing.

Effects of dietary crude protein and rumen-degradable protein concentrations on urea recycling, nitrogen balance, omasal nutrient flow, and milk production in dairy cows.

The objective of this study was to determine how interactions between dietary crude protein (CP) and rumen-degradable protein (RDP) concentrations alter urea-nitrogen recycling, nitrogen (N) balance, omasal nutrient flow, and milk production in lactating Holstein cows. Eight multiparous Holstein cows (711±21kg of body weight; 91±17d in milk at the start of the experiment) were used in a replicated 4×4 Latin square design with a 2×2 factorial arrangement of dietary treatments and 29-d experimental periods. Four cows in one Latin square were fitted with ruminal cannulas to allow ruminal and omasal sampling. The dietary treatment factors were CP (14.9 vs. 17.5%; dry matter basis) and RDP (63 vs. 69% of CP) contents. Dietary RDP concentration was manipulated by including unprocessed or micronized canola meal. Diet adaptation (d 1-20) was followed by 8d (d 21-29) of sample and data collection. Continuous intrajugular infusions of [(15)N(15)N]-urea (220mg/d) were conducted for 4d (d 25-29) with concurrent total collections of urine and feces to estimate N balance and whole-body urea kinetics. Proportions of [(15)N(15)N]- and [(14)N(15)N]-urea in urinary urea, and (15)N enrichment in feces were used to calculate urea kinetics. For the low-CP diets, cows fed the high-RDP diet had a greater DM intake compared with those fed the low-RDP diet, but the opposite trend was observed for cows fed the high-CP diets. Dietary treatment had no effect on milk yield. Milk composition and milk component yields were largely unaffected by dietary treatment; however, on the low-CP diets, milk fat yield was greater for cows fed the low-RDP diet compared with those fed the high-RDP diet, but it was unaffected by RDP concentration on the high-CP diets. On the high-CP diets, milk urea nitrogen concentration was greater in cows fed the high-RDP diet compared with those fed the low-RDP diet, but it was unaffected by RDP concentration on the low-CP diets. Ruminal NH3-N concentration tended to be greater in cows fed the high-CP diet compared with those fed the low-CP diet, and it was greater in cows fed the high-RDP diet as compared with those fed the low-RDP diet. Nitrogen intake and both total N and urea-N excretion in urine were greater for cows fed the high-CP diet compared with those fed the low-CP diet. However, N balance and urinary excretion of purine derivatives were unaffected by dietary treatment. Urea-N entry rate (UER) was greater in cows fed the high-CP diet compared with those fed the low-CP diet; however, UER was unaffected by dietary RDP concentration. The proportion of urea-N recycled to the gastrointestinal tract (as a percentage of UER) was greater in cows fed the low-CP diet compared with those fed the high-CP diet. In summary, reducing dietary CP concentration decreased urinary N excretion but had no effect on milk yield, thus resulting in an overall improvement in milk N efficiency.

Early results of a prospective study on the pyrolytic carbon (pyrocarbon) Amandys® for osteoarthritis of the wrist.

INTRODUCTION: The preliminary results of a pyrocarbon interpositional radiocarpal implant in a small cohort of patients were reviewed. As it is currently only a limited release product, we describe to potential users early complications and negative outcomes. METHODS: Patients were assessed using pain levels, ranges of motion, grip strength, type of and time to return to work as well as pre-operative and post-operative DASH (Disabilities of the Arm, Shoulder and Hand) scores. Radiographs were taken and patient satisfaction was recorded. RESULTS: All six patients were contacted. One was not satisfied. Three had reduced motion. None experienced squeaking. There were no immediate or late post-operative complications. There was one early volar displacement of an implant. CONCLUSIONS: Although our early results are somewhat encouraging, further and longer studies are warranted before supporting the use of this particular pyrocarbon implant as a primary procedure.

Characteristics of peaks in serum concentrations of follicle-stimulating hormone and estradiol, and follicular wave dynamics during the interovulatory interval in cyclic ewes.

There are three or four ovarian follicular waves in the interovulatory interval of cyclic ewes. Each follicular wave is preceded by a transient peak in serum follicle-stimulating hormone (FSH) concentrations. Serum concentrations of estradiol also increase concurrent with the growth of follicle(s) in each wave. In the current study, we investigated the patterns of follicular wave development and characteristics of FSH and estradiol peaks in all follicular waves of the interovulatory interval and after induction of a supraphysiologic FSH peak in cyclic ewes (Ovis aris). In Experiment 1, 19 ewes underwent daily ovarian ultrasonography and blood sampling for a complete interovulatory interval. In Experiment 2, seven ewes received two administrations of ovine FSH (oFSH), 8h apart (1 microg/kg; sc), at the expected time of the endogenous FSH peak preceding the second follicular wave of the interovulatory interval. In Experiment 1, the amplitude of the FSH peaks decreased (up to 50%), whereas basal serum FSH concentrations increased across the interovulatory interval (P<0.05). Maximum follicular diameter was greater (P<0.05) for Wave 1 and the Ovulatory wave (6.0+/-0.3 and 6.1+/-0.2 mm, respectively) than for Waves 2 and 3 (5.3+/-0.1 and 5.4+/-0.3 mm, respectively). Life span was greater for follicles in Wave 1 compared with other waves (P<0.05). Treatment with oFSH increased the amplitude of an FSH peak by 5- to 6-fold. This treatment increased estradiol production (P<0.05) but had little effect on other characteristics of the subsequent follicular wave. We concluded that changes in the amplitude and duration of the peaks in serum concentrations of FSH that precede follicular waves across the interovulatory interval do not influence the characteristics of the follicular waves that follow.

Ovarian antral follicular dynamics in sheep revisited: comparison among estrous cycles with three or four follicular waves.

In this study, the characteristics of ovarian follicular waves and patterns of serum concentrations of follicle-stimulating hormone (FSH), estradiol, and progesterone were compared between cycles with three (n=9) or four (n=10) follicular waves in Western White Face (WWF) ewes (Ovis aries). Transrectal ultrasonography and blood sampling were performed daily during one cycle. Estrous cycles were 17.11+/-0.3 and 17.20+/-0.2 d long in cycles with three and four waves, respectively (P>0.05). The first interwave interval and the interval from the emergence of the final wave to the day of ovulation were longer in cycles with three waves compared with those in cycles with four waves (P<0.05). The growth phase (5.1+/-0.5 vs. 3.1+/-0.4 d) and life span (5.67+/-0.3 vs. 4.3+/-0.3 d) of the largest follicle growing in the last or ovulatory wave was longer in cycles with three waves compared with that in cycles with four waves (P<0.05). The maximum diameter of the largest follicle was greater in the first wave and the ovulatory wave compared with that in other waves of the cycle (P<0.05). The regression phase of the largest follicle growing in the first wave was longer in cycles with three waves compared with that in cycles with four waves (4.44+/-0.4 vs. 3.4+/-0.4 d; P<0.05). The length of the life span, regression phase, and, although not significant in every case, FSH peak concentration and amplitude decreased across the cycle (P<0.05). We concluded that estrous cycles with three or four follicular waves were confined within the same length of cycle in WWF ewes. In this study, there were no apparent endocrine or follicular characteristics that could explain the regulation of the different number of follicular waves (three vs. four) during cycles of similar length.

The effect of parity of the dam on sexual maturation, serum concentrations of metabolic hormones and the response to luteinizing hormone releasing hormone in bull calves.

The objectives of this study were to determine the effect of parity of the dam on age at which a scrotal circumference (SC) ≥ 28 cm was attained and the LH response to Luteinizing Hormone Releasing Hormone (LHRH) in bull calves. We also wanted to confirm, in a large group of bull calves, that the LH response to LHRH could be used to select early maturing bulls. Body weight and SC of the bull calves were measured every other week. At 15, 20 and 25 weeks of age, calves received 4.12 nm/kg body weight of LHRH ischio-rectally and blood samples were taken every 15 min for 4 h. Calves from primiparous and multiparous dams were separated into two sub-groups based on age at which an SC ≥ 28 cm was attained (early or late). An SC ≥ 28 cm was attained earlier in calves born to multiparous as compared with primiparous dams (p < 0.05). At 20 and 25 weeks of age, peak serum LH concentrations (LH-peak) and area under the LH response curve (LH-AUC) in response to LHRH were higher (p < 0.01) in calves born to multiparous as compared with primiparous dams. In calves born to multiparous dams the LH-peak at 15 and 25 weeks of age and the LH-AUC at 15 weeks of age were lower (p < 0.05) in calves that attained an SC ≥ 28 cm early as compared with late. The LHRH-challenge test sensitivity and specificity ranged from 46% to 86%. We concluded that parity of the dam affected age at which SC ≥ 28 cm was attained and the LH response to LHRH in bull calves. Serum LH responses to LHRH at 15 and 25 weeks of age, in calves born to multiparous dams, show some promise for development into a test to select early maturing bull calves.

Synchronization of follicular wave emergence in the seasonally anestrous ewe: the effects of estradiol with or without medroxyprogesterone acetate.

Fertility is often lower in anestrous compared to cyclic ewes, after conventional estrus synchronization. We hypothesized that synchronization of ovarian follicular waves and ovulation could improve fertility at controlled breeding in anestrous ewes. Estradiol-17beta synchronizes follicular waves in cattle. The objectives of the present experiments were to study the effect of an estradiol injection, with or without a 12-d medroxyprogesterone acetate (MAP) sponge treatment, on synchronization of follicular waves and ovulation in anestrous ewes. Twenty ewes received sesame oil (n=8) or estradiol-17beta (350 microg; n=12). Eleven ewes received MAP sponges for 12d and were treated with oil (n=5) or estradiol-17beta (n=6) 6d before sponge removal. Saline (n=6) or eCG (n=6) was subsequently given to separate groups of ewes at sponge removal in the MAP/estradiol-17beta protocol. Estradiol treatment alone produced a peak in serum FSH concentrations (4.73+/-0.53 vs. 2.36+/-0.39 ng/mL for treatment vs. control; mean+/-S.E.M.) after a short-lived (6 h) suppression. Six of twelve ewes given estradiol missed a follicular wave around the time of estradiol injection. Medroxyprogesterone acetate-treated ewes given estradiol had more prolonged suppression of serum FSH concentrations (6-18 h) and a delay in the induced FSH peak (32.3+/-3.3 vs. 17.5+/-0.5 h). Wave emergence was delayed (5.7+/-0.3 vs. 1.4+/-0.7d from the time of estradiol injection), synchronized, and occurred at a predictable time (5-7 vs. 0-4d) compared to ewes given MAP alone. All ewes given eCG ovulated 3-4d after injection; this predictable time of ovulation may be efficacious for AI and embryo transfer.

Labellar micromorphology of Bifrenariinae Dressler (Orchidaceae).

BACKGROUND AND AIMS: The two closely related subtribes Bifrenariinae Dressler and Maxillariinae Benth. are easily distinguished on morphological grounds. Recently, however, molecular techniques have supported the inclusion of Bifrenariinae within a more broadly defined Maxillariinae. The present paper describes the diverse labellar micromorphology found amongst representatives of Bifrenariinae (Bifrenaria Lindl., Rudolfiella Hoehne, Teuscheria Garay and Xylobium Lindl.) and compares it with that found in Maxillaria Pabst & Dungs and Mormolyca Fenzl (Maxillariinae). METHODS: The labella of 35 specimens representing 22 species of Bifrenariinae were examined by means of light microscopy and scanning electron microscopy and their micromorphology compared with that of Maxillaria sensu stricto and Mormolyca spp. The labellar epidermis of representatives of Bifrenaria, Xylobium and Mormolyca was tested for protein, starch and lipids in order to ascertain whether this tissue is involved in the rewarding of pollinators. KEY RESULTS AND CONCLUSIONS: The labella of Bifrenaria spp. and Mormolyca spp. are densely pubescent but those of Xylobium, Teuscheria and Rudolfiella are generally papillose. However, whereas the trichomes of Bifrenaria and Mormolyca are unicellular, those found in the other three genera are multicellular. Hitherto, no unicellular trichomes have been described for Maxillaria, although the labella of a number of species secrete a viscid substance or bear moniliform, pseudopollen-producing hairs. Moniliform hairs and secretory material also occur in certain species of Xylobium and Teuscheria and these genera, together with Maxillaria, are thought to be pollinated by stingless bees (Meliponini). Differences in the labellar micromorphology of Bifrenaria and Mormolyca are perhaps related to Euglossine- and/ or bumble bee-mediated pollination and pseudocopulation, respectively. Although Xylobium and Teuscheria share a number of labellar features with Maxillaria sensu stricto, this does not necessarily reflect taxonomic relationships but may be indicative of convergence in response to similar pollinator pressures.

A Rudimentary labellar speculum in Cymbidium lowianum (Rchb.f.) Rchb.f. and Cymbidium devonianum Paxton (Orchidaceae).

BACKGROUND AND AIMS: The labellar 'hairs' of some Cymbidium spp. are said to be thin-walled and to contain 'plasma', oil and sugars and it has long been speculated that they may function as food-hairs. However, the present authors' preliminary studies showed that certain atypical papillae may have a different role and, by reflecting light, function as a speculum. The purpose of the paper is to test this hypothesis. METHODS: Light microscopy, scanning electron microscopy, transmission electron microscopy, histochemistry and ultraviolet photography were used to investigate the structure, food content and light-reflecting properties of these papillae. KEY RESULTS AND CONCLUSIONS: The labellum of Cymbidium lowianum (Rchb.f.) Rchb.f. is densely clothed with obconical to conical papillae with wide bases and pointed tips. However, on either side of the median axis of the lip occur silvery patches comprising papillae with truncated tips and it is thought that these reflect light and thereby attract insect pollinators. Similar patches are also found in Cymbidium devonianum Paxton, and in both species, they are set against a reddish background, which, since bees cannot perceive this colour, probably appears dark to the insect thus enhancing the visual impact of the light-reflecting patches. In Cymbidium tigrinum Parish ex Hook. and Cymbidium mastersii Griff. ex Lindl., however, the labellum is mainly white and no light-reflecting patches were observed. Instead, unlike C. lowianum and C. devonianum, these species are highly fragrant and the attraction of insects probably depends to a greater extent on olfactory cues. In C. lowianum both types of papillae contain protein, starch and lipid bodies but only protein is seemingly present at elevated concentrations. However, lipoidal material also occurs upon the surface of the labellum and it is possible that this may be gathered by insects as reported for C. iridifolium A. Cunn (syn. C. madidum Lindl.). The labellar papillae of C. lowianum, thus, have the potential to function as food-hairs, although direct evidence for this is lacking.

Computer assisted image analyses of corpora lutea in relation to peripheral concentrations of progesterone: a comparison between breeds of sheep with different ovulation rates.

Transrectal ovarian ultrasonography is a non-invasive technique that permits the real-time serial visualization of ovarian structures. Previous studies have reported correlations between serum progesterone concentrations and physical characteristics of the corpus luteum (CL) in different species. The aim of the present study was to investigate whether or not there were correlations between ultrasound image attributes of the CL and changing progesterone concentrations over time, in prolific and non-prolific ewes. Ultrasonographic images of CL were taken, once daily, from 12 Western White Face ewes and 7 Finn ewes for the duration of one luteal phase. Blood samples were collected daily prior to each scanning session and assayed to determine mean serum concentrations of progesterone. Analysis of ultrasound images was performed using a series of custom-developed computer algorithms optimized for ultrasonography on a computer graphics workstation. Both total luteal area and mean pixel values were correlated with the pattern of serum concentrations of progesterone from days 3 to 15 after ovulation in Western White Face ewes and from days 3 to 14 in Finn ewes. There was no significant correlation between progesterone concentrations and spot pixel heterogeneity for either Western White Face ewes or Finn ewes. We concluded that pixel heterogeneity is a poor indicator of progesterone secretory ability of the CL when compared to mean pixel values. However, luteal area and mean spot pixel values are better but not strong indicators of the functional status of the CL in cyclic ewes.

Patterns of expression of steroidogenic enzymes during the first wave of the ovine estrous cycle as compared to the preovulatory follicle.

The expression patterns of steroidogenic enzymes in ovarian antral follicles at various stages of growth in a follicular wave have not been reported for sheep. Ovaries were collected from ewes (n=4-5 per group) when the largest follicle(s) of the first wave of the cycle, as determined by ultrasonography, reached (i) 3 mm, (ii) 4 mm, (iii) > or =5 mm in diameter or when there was a single (iv) preovulatory follicle in the last wave of the cycle, 12h after estrus detection. The expression pattern of steroidogenic enzymes was quantified using immunohistochemistry and grey-scale densitometry. The expression of CYP19 in the granulosa and 3beta-HSD and CYP17 in the theca increased (P<0.01) progressively from 3 to > or =5 mm follicles in the first wave of the cycle and was lower (P<0.01) in the preovulatory follicle compared to > or =5 mm follicles. However, the expression of 3beta-HSD in the granulosa increased (P<0.05) from 3 to > or =5 mm follicles and was maintained (P<0.05) at a high level in the preovulatory follicles. The amount of CYP19 in the granulosa of the growing follicles correlated positively (r=0.5; P<0.03) with the concurrent serum estradiol concentrations. We concluded that the expression pattern of steroidogenic enzymes in theca and granulosa of follicles growing in each wave in the ewe, paralleled with serum estradiol concentrations, with the exception that concentrations of 3beta-HSD in granulosa increased continuously from follicles 3mm in diameter to the preovulatory follicle.

Does injection of prostaglandin F(2alpha) (PGF2alpha) cause ovulation in anestrous Western White Face ewes?

In a previous study in our laboratory, treatment of non-prolific Western White Face (WWF) ewes with PGF(2 alpha) and intravaginal sponges containing medroxyprogesterone acetate (MAP) on approximately Day 8 of a cycle (Day 0 = first ovulation of the interovulatory interval) resulted in ovulations during the subsequent 6 days when MAP sponges were in place. Two experiments were performed on WWF ewes during anestrus to allow us to independently examine if such ovulations were due to the direct effects of PGF(2 alpha) on the ovary or to the effects of a rapid decrease in serum concentrations of progesterone at PGF(2 alpha)-induced luteolysis. Experiment 1: ewes fitted with MAP sponges for 6 days (n = 12) were injected with PGF(2 alpha) (n = 6; 15 mg im), or saline (n = 6) on the day of sponge insertion. Experiment 2: ewes received progesterone-releasing subcutaneous implants (n = 6) or empty implants (n = 5) for 5 days. Six hours prior to implant removal, all ewes received a MAP sponge, which remained in place for 6 days. Ewes from both experiments underwent ovarian ultrasonography and blood sampling once daily for 6 days before and twice daily for 6 days after sponge insertion. Additional blood samples were collected every 4 h during sponge treatment. Experiment 1: 4-6 (67%) PGF(2 alpha)-treated ewes ovulated approximately 1.5 days after PGF(2 alpha) injection; these ovulations were not preceded by estrus or a preovulatory surge release of LH, and resulted in transient corpora hemorrhagica (CH). The growth phase was longer (P < 0.05) and the growth rate slower (P < 0.05) in ovulating versus non-ovulating follicles in PGF(2 alpha)-treated ewes. Experiment 2: in ewes given progesterone implants, serum progesterone concentrations reached a peak (1.7 2 ng/mL; P < 0.001) on the day of implant removal and decreased to basal concentrations (<0.17 ng/mL; P < 0.001) within 24 h of implant removal. No ovulations occurred in either the treated or the control ewes. We concluded that ovulations occurring after PGF(2 alpha) injection, in the presence of a MAP sponge, could be due to a direct effect of PGF(2 alpha) at the ovarian level, rather than a sudden decline in circulating progesterone concentrations.

Nectar-secreting floral stomata in Maxillaria anceps Ames & C. Schweinf. (Orchidaceae).

BACKGROUND AND AIMS: Although it was generally assumed that Maxillaria spp. do not produce nectar, in recent years, nectar has been reported for a number of these orchids. Nevertheless, our current understanding of nectary structure and nectar secretion in Maxillaria is based solely on M. coccinea (Jacq.) L.O. Williams ex Hodge, which, since it shows many features characteristic of ornithophilous flowers, is atypical of this largely entomophilous genus. The aim of the present paper is to describe, for the first time, nectar secretion in a presumed entomophilous species of Maxillaria. METHODS: The structure of the nectary of M. anceps Ames & C. Schweinf., nectar composition and the process of nectar secretion were investigated using light microscopy, scanning electron microscopy, transmission electron microscopy, histochemistry, refractometry and high performance liquid chromatography. KEY RESULTS AND CONCLUSIONS: Nectar appears as droplets that are exuded by modified stomata borne upon the labellar callus and collects upon the labellum and at the base of the column-foot. Although such stomata are known to occur in a number of angiosperm families, this is the first time for them to be observed in orchids. The callus consists largely of parenchyma with raphides and is supplied by eight to ten collateral bundles. This tissue, together with the single-layered epidermis, seemingly contains terpenoids. During the bud stage, the callus cells contain an organelle complement consistent with secretory cells whereas by day 4 of anthesis, much of the cell is occupied by a vacuole. The nectar is sucrose-dominant but also contains low concentrations of glucose, fructose, free amino acids and possibly terpenoids. The high sugar concentration (approx. 66 %) is consistent with melittophily and may indicate that, like the majority of Maxillaria spp., M. anceps is visited by stingless bees (Meliponini).

Comparative account of nectary structure in Hexisea imbricata (Lindl.) Rchb.f. (Orchidaceae).

BACKGROUND AND AIMS: Despite the number of orchid species that are thought to be pollinated by hummingbirds, our knowledge of the nectaries of these orchids is based solely on a single species, Maxillaria coccinea (Jacq.) L.O. Williams ex Hodge. Nevertheless, it is predicted that such nectaries are likely to be very diverse and the purpose of this paper is to compare the nectary and the process of nectar secretion in Hexisea imbricata (Lindl.) Rchb.f. with that of Maxillaria coccinea so as to begin to characterize the nectaries of presumed ornithophilous Neotropical orchids. METHODS: Light microscopy, transmission electronmicroscopy and histochemistry were used to examine the histology and chemical composition of nectary tissue and the process of nectar secretion in H. imbricata. KEY RESULTS AND CONCLUSIONS: The nectary of H. imbricata has a vascular supply, is bound by a single-layered epidermis with few stomata and comprises two or three layers of subepidermal secretory cells beneath which lie several layers of palisade-like parenchymatous cells, some of which contain raphides or mucilage. The secretory cells are collenchymatous and their walls have numerous pits with associated plasmodesmata. They contain the full complement of organelles characteristic of secretory cells as well as intravacuolar protein bodies but some of the secretory epidermal cells, following secretion, collapse and their anticlinal walls seem to fold. Nectar secretion is thought to be granulocrine and, following starch depletion, lipid droplets collect within the plastids. The nectar accumulates beneath the cuticle which subsequently forms swellings. Finally, nectar collects in the saccate nectary spur formed by the fusion of the margins of the labellum and the base of the column-foot. Thus, although the nectary of H. imbricata and M. coccinea have many features in common, they nevertheless display a number of important differences.

Pseudopollen in Eria Lindl. section Mycaranthes Rchb.f. (Orchidaceae).

BACKGROUND AND AIMS: Pseudopollen is a whitish, mealy material produced upon the labella of a number of orchid species as labellar hairs either become detached or fragment. Since individual hair cells are rich in protein and starch, it has long been speculated that pseudopollen functions as a reward for visiting insects. Although some 90 years have passed since Beck first described pseudopollen for a small number of Eria spp. currently assigned to section Mycaranthes Rchb.f., we still know little about the character of pseudopollen in this taxon. The use of SEM and histochemistry would re-address this deficit in our knowledge whereas comparison of pseudopollen in Eria (S.E. Asia), Maxillaria (tropical and sub-tropical America), Polystachya (largely tropical Africa and Madagascar) and Dendrobium unicum (Thailand and Laos) would perhaps help us to understand better how this feature may have arisen and evolved on a number of different continents. METHODS: Pseudopollen morphology is described using light microscopy and scanning electron microscopy. Hairs were tested for starch, lipid and protein using IKI, Sudan III and the xanthoproteic test, respectively. KEY RESULTS AND CONCLUSIONS: The labellar hairs of all eight representatives of section Mycaranthes examined are identical. They are unicellular, clavate with a narrow 'stalk' and contain both protein and starch but no detectable lipid droplets. The protein is distributed throughout the cytoplasm and the starch is confined to amyloplasts. The hairs become detached from the labellar surface and bear raised cuticular ridges and flaky deposits that are presumed to be wax. In that they are unicellular and appear to bear wax distally, the labellar hairs are significantly different from those observed for other orchid species. Comparative morphology indicates that they evolved independently in response to pollinator pressures similar to those experienced by other unrelated pseudopollen-forming orchids on other continents.

Pseudopollen in Dendrobium unicum Seidenf. (Orchidaceae): reward or deception?

BACKGROUND AND AIMS: In 1987, Kjellsson and Rasmussen described the labellar trichomes of Dendrobium unicum Seidenf. and proposed that these hairs function as pseudopollen. Pseudopollen is a mealy material that superficially resembles pollen, is usually laden with food substances and is formed when labellar hairs either fragment into individual cells or become detached from the labellum. However, the trichomes of D. unicum are very different from pseudopollen-forming hairs found in other orchid genera such as Maxillaria and Polystachya. Moreover, Kjellsson and Rasmussen were unable to demonstrate the presence of food substances within these trichomes and argued that even in the absence of food substances, the hairs, in that they superficially resemble pollen, can still attract insects by deceit. The aim of this paper is to investigate whether the labellar trichomes of D. unicum contain food reserves and thus reward potential pollinators or whether they are devoid of foods and attract insects solely by mimicry. METHODS: Light microscopy, histochemistry and transmission electron microscopy. KEY RESULTS: Dendrobium unicum produces pseudopollen. Pseudopollen here, however, differs from that previously described for other orchid genera in that the pseudopollen-forming trichomes consist of a stalk cell and a 'head' of component cells that separate at maturity, in contrast to Maxillaria and some Polystachya spp. where pseudopollen is formed by the fragmentation of moniliform hairs. Moreover, the pseudopollen of Maxillaria and Polystachya largely contains protein, whereas in D. unicum the main food substance is starch. CONCLUSIONS: Flowers of D. unicum, rather than attracting insects solely by deceit may also reward potential pollinators.