Evaluation of a commercially available radioimmunoassay and enzyme immunoassay for the analysis of progesterone and estradiol and the comparison of two extraction efficiency methods.

The measurement of progesterone (P4) and estradiol (E2) is essential for monitoring reproductive cycles and can aid in diagnosing the cause of poor reproductive performance in dairy cattle. Readily available, reproducible, accurate, non-radioactive assays are needed for the assessment of P4 and E2 in bovine serum. The gold standard for hormone assessment, radioimmunoassay (RIA), was compared with enzyme-linked immunoassay (EIA). Serum collected from various points in the estrous cycle was extracted with radiolabeled P4 (ie, 3H-P4; HE) and without 3H-P4 (CE) before being used in the assay. For the assessment of P4, there is a great degree of correlation between the RIA and EIA (adjusted R-square = 0.95; Pearson correlation coefficient (PCC) = 0.98, P < 0.001). A difference between the RIA and EIA methods was not detected for E2 concentrations (P = 0.16), but the correlation between techniques was poor (adjusted R-squared = 0.73; PCC = 0.87, P = 0.002). There was no difference in the serum extraction efficiency as measured with 3H-P4 as opposed to without (P = 0.94). The two methods for the measurement of serum extraction efficiency were highly correlated (adjusted R-square = 0.83; PCC = 0.92, P < 0.001). The concordance correlation coefficient (CCC) showed an excellent agreement between RIA and EIA for P4 determination (0.89) and between HE and CE methods (0.90). Although the 95% limits of agreement of the Bland-Altman plots encompassed 89% (8/9) and 92% (12/13) of the differences between methods for P4 quantification and extraction respectively, the CCC indicated an excellent agreement among them. The CCC between RIA and EIA for E2 quantification was 0.68 which corresponds with a fair agreement; however, the 95% limits of agreement of the Bland-Altman plot encompassed 100% (9/9) of differences between methods. The EIA and CE methods are comparable alternatives to the RIA and HE methods, respectively and can be used to quantify P4 and E2 for bovine serum.

Vaccination against the avian infectious bronchitis virus affects sperm concentration, sperm quality and blood testosterone concentrations in cockerels.

1. It was previously found that cockerels vaccinated with live attenuated avian infectious bronchitis virus (AIBV) have decreased serum testosterone concentrations, epididymal stones and reduced fertility. The objectives of this study were twofold: to determine if reduced fertility following vaccination with live attenuated virus was the result of reduced sperm concentration or reduced sperm quality and to determine if vaccination with a killed strain of virus caused a similar reduction in sperm function in vivo. 2. Specific-pathogen-free Single Comb White Leghorn cockerels were divided into three treatment groups: no vaccination (NONVAC), vaccination with killed AIBV virus (KVAC) or vaccination with live attenuated AIBV virus (LVAC). Semen was collected daily from 17 to 27 weeks of age, and semen quality was assessed frequently by analysing sperm concentration, viability, motility, and ability to reach and interact with the ovum in vivo. Blood plasma was assayed for testosterone concentration. 3. Differences in sperm analysis among treatment groups were limited. Sperm viability was increased in NONVAC during week 20 which then decreased in week 22 when compared to vaccinated cockerels. Acrosome damage was increased in vaccinated cockerels in week 22, and decreased in weeks 25 and 27 when compared to controls, which correlate to the period of epididymal stone development. Plasma testosterone concentrations and sperm concentrations among treatment groups were different only at 16 and 19 weeks of age, respectively. There were no differences across treatment groups in sperm mobility through Accudenz or in numbers of sperm holes in perivitelline membranes of eggs following insemination with semen from 27-week-old cockerels. No differences were observed in viability or acrosome integrity between cockerels with and without epididymal stones within treatment groups. 4. In conclusion, pre-pubertal vaccination against AIBV and subsequent epididymal stone formation had a limited effect on sperm concentration, sperm quality and plasma testosterone concentrations. Vaccination with killed AIBV vaccine did not diminish effects on sperm function in vivo.

Ion transporters for fluid reabsorption in the rooster (Gallus domesticus) epididymal region.

Testicular fluid is highly condensed during its passage through the epididymal region in the avian species. In the present study, major ion transporters that are responsible for condensation mainly by water resorption in the reproductive tract as identified in the mammalian epididymis were localized within the rooster (Gallus domesticus) epididymis by immunohistochemistry. The results show that the efferent ductule epithelium expressed sodium-potassium ATPase (Na(+),K(+)-ATPase), carbonic anhydrase II (CAII) and sodium hydrogen exchanger isoform 3 (NHE3) and that the connecting ductule and epididymal duct epithelia expressed Na(+),K(+)-ATPase and CAII. These data suggest that a model proposed for reabsorption in mammalian efferent ductules can be applied to avian efferent ductules.

Stages and duration of spermatogenesis in the domestic ferret (Mustela putorius furo).

Classification of seminiferous tubules is the basis for understanding normal and abnormal spermatogenesis. The aim of the present study was to determine spermatogenic stages and the duration of the cycle in the domestic ferret using bromodeoxyuridine (BrdU) as a tracer. Eleven adult male ferrets that were maintained in a breeding condition were used. Testicular sections were stained with the periodic acid-Schiff reaction for light microscopy. To determine the cycle duration, six ferrets were injected intraperitoneally with BrdU, and testes were collected 3h later and 10 days and 3h later. BrdU was detected by immunohistochemistry. Seminiferous tubules were classified into eight stages, and frequencies of stages I-VIII were 10.6, 2.2, 7.9, 13.1, 22.3, 21.9, 14.0 and 8.0%, respectively. The most advanced BrdU-labeled cells at 3h post-injection were leptotene spermatocytes in stage VI and those at 10 days and 3h were pachytene spermatocytes in stage V. From differences in stage frequency and BrdU staining frequency between two time points, the duration of one cycle was estimated to be 13.0 days. The present observations indicate that stages and the cycle duration of the ferret spermatogenesis are similar to those reported in other carnivores.

Mechanisms of electron acceptor utilization: implications for simulating anaerobic biodegradation.

Simulation of biodegradation reactions within a reactive transport framework requires information on mechanisms of terminal electron acceptor processes (TEAPs). In initial modeling efforts, TEAPs were approximated as occurring sequentially, with the highest energy-yielding electron acceptors (e.g. oxygen) consumed before those that yield less energy (e.g., sulfate). Within this framework in a steady state plume, sequential electron acceptor utilization would theoretically produce methane at an organic-rich source and Fe(II) further downgradient, resulting in a limited zone of Fe(II) and methane overlap. However, contaminant plumes often display much more extensive zones of overlapping Fe(II) and methane. The extensive overlap could be caused by several abiotic and biotic processes including vertical mixing of byproducts in long-screened monitoring wells, adsorption of Fe(II) onto aquifer solids, or microscale heterogeneity in Fe(III) concentrations. Alternatively, the overlap could be due to simultaneous utilization of terminal electron acceptors. Because biodegradation rates are controlled by TEAPs, evaluating the mechanisms of electron acceptor utilization is critical for improving prediction of contaminant mass losses due to biodegradation. Using BioRedox-MT3DMS, a three-dimensional, multi-species reactive transport code, we simulated the current configurations of a BTEX plume and TEAP zones at a petroleum-contaminated field site in Wisconsin. Simulation results suggest that BTEX mass loss due to biodegradation is greatest under oxygen-reducing conditions, with smaller but similar contributions to mass loss from biodegradation under Fe(III)-reducing, sulfate-reducing, and methanogenic conditions. Results of sensitivity calculations document that BTEX losses due to biodegradation are most sensitive to the age of the plume, while the shape of the BTEX plume is most sensitive to effective porosity and rate constants for biodegradation under Fe(III)-reducing and methanogenic conditions. Using this transport model, we had limited success in simulating overlap of redox products using reasonable ranges of parameters within a strictly sequential electron acceptor utilization framework. Simulation results indicate that overlap of redox products cannot be accurately simulated using the constructed model, suggesting either that Fe(III) reduction and methanogenesis are occurring simultaneously in the source area, or that heterogeneities in Fe(III) concentration and/or mineral type cause the observed overlap. Additional field, experimental, and modeling studies will be needed to address these questions.

Role of FSH and epidermal growth factor (EGF) in the initiation of steroidogenesis in granulosa cells associated with follicular selection in chicken ovaries.

In chicken ovaries, one small yellow follicle (SYF) is selected daily from a pool of follicles of similar size and becomes a preovulatory follicle. FSH induces follicular growth and steroidogenesis. Epidermal growth factor (EGF), an intraovarian hormone, suppresses granulosa cell differentiation. This study demonstrates that recruitment of SYFs into the hierarchy of preovulatory follicles is associated with a change in steroidogenic activity in granulosa cells regulated, at least in part, by FSH and EGF. Abundance of P450 side-chain cleavage (P450scc) mRNA was higher in the smallest preovulatory follicle (F6) compared with SYF, whereas FSH and EGF receptor (FSHr and EGFr, respectively) mRNA abundance was similar. FSH increased P450scc mRNA abundance and progesterone secretion and decreased FSHr mRNA in cultured granulosa cells, whereas EGF attenuated or suppressed P450scc mRNA and decreased FSHr mRNA abundance. None of the hormones influenced EGFr mRNA abundance. When used in combination, EGF attenuated or suppressed the stimulatory effect of FSH on the expression of P450scc mRNA and production of progesterone in a dose-dependent manner. The results indicate that (1) selection is associated with an increase in P450scc mRNA; (2) FSH stimulates expression of P450scc mRNA and progesterone secretion in granulosa cells of SYF; and (3) induction of P450scc mRNA and progesterone secretion by FSH is attenuated or blocked by EGF.

Follicular development and steroid concentrations in cows with different levels of fertility raised under nutritional stress.

The aim of the present study was to characterize ovarian follicular development and steroid concentrations during postpartum and the estrous cycle of Brangus Ibagé cows (3/8 Nelore + 5/8 Aberdeen Angus) with different levels of fertility. Cows were classified as having high or low fertility according to the calving interval (CI). The average CI of the herd from which cows used in this study were selected was 404.6+/-5.44 and 711.2+/-20.89 days for the high and low fertility groups, respectively. Four cows of high fertility and five cows of low fertility had calves removed between 70 and 100 days after parturition. Ovarian activity was monitored daily by ultrasound for 16 days after calf removal. Days to emergency of the first follicular wave after calf removal, number of follicles with diameter >9 mm, growth rate of largest follicle, maximum diameter of largest follicle, length (days) and number of follicular waves were recorded. During this period, blood was collected daily for measurements of serum progesterone (P(4)) and estradiol (E(2)) concentrations. In another experiment, ovarian activity and P(4) and E(2) concentrations were examined during estrous cycle in five cows of high fertility and four cows of low fertility. Ovarian activity and steroid concentrations were assessed from the day prior to estrus to the 15th day of the estrous cycle (estrus = day 0). In postpartum cows of high fertility, the total number of follicles >5mm and the maximum diameter of the largest follicle were higher than in cows of low fertility (P < 0.05). Concentrations of P(4) and E(2) did not differ between groups in the postpartum cows. However, E(2) increased 5 days after calf removal (around 90 days of postpartum) in the high fertility group, followed by an increase in P(4) with average values indicating ovulation around 100 days postpartum. In cycling cows, the profile of follicular development was similar between cows of high and low fertility. There was no difference between groups for number of follicles >5mm, but the day effect was significant (P < 0.01). Plasma concentrations of P(4) and E(2) were similar in both groups. These data suggest that cows, from a population raised in the same environment have different fertility as a consequence of individual physiological characteristics.

Seasonal changes in testicular size and serum LH, prolactin and testosterone concentrations in male polar bears (Ursus maritimus).

Little is known about the reproductive endocrinology of the male polar bear, Ursus maritimus, except that serum testosterone concentrations are high in April and May during the mating season and are low from August to November during the non-mating season. The objective of this study was to describe the relationship between seasonal changes in testicular size and serum concentrations of testosterone, LH and prolactin. Blood samples and testicular measurements were obtained from free-ranging male polar bears in Canada in April (n = 5) and May (n = 15) near Resolute Bay, Northwest Territories and near Churchill, Manitoba in July (n = 15) and October (n = 22). Testis size was greater in May (39.4 +/- 3.5 cm(2)) than in October (27.3 +/- 2.0 cm(2)) (P = 0.002). Serum testosterone concentrations were approximately three-fold higher in April (5.8 +/- 0.8 ng ml(-1)) than in May (1.7 +/- 0.5 ng ml(-1)), July (0.6 +/- 0.2 ng ml(-1)) and October (1.1 +/- 0.2 ng ml(-1)). Similarly, serum LH concentrations were high in April (0.14 +/- 0.04 ng ml(-1)) and low in May (0.09 +/- 0.01 ng ml(-1)), July (0.10 +/- 0.02 ng ml(-1)) and October (0.08 +/- 0.00 ng ml(-1)). Serum prolactin concentrations were high in April (1.9 +/- 0.3 ng ml(-1)), highest in May (2.5 +/- 0.2 ng ml(-1)), lower in July (1.3 +/- 0.1 ng ml(-1)) and lowest in October (0.8 +/- 0.07 ng ml(-1)). The present study demonstrates a positive relationship between testicular size and serum concentrations of LH, prolactin and testosterone in the male polar bear and confirms the previously reported seasonal changes in serum testosterone concentrations. Data from the present study provide important baseline and comparative endocrine information that can be used to aid captive breeding programmes in zoos and to further ecological-behavioural studies of polar bears.

Nitrate-enhanced bioremediation of BTEX-contaminated groundwater: parameter estimation from natural-gradient tracer experiments.

Two natural-gradient pulse tracer tests were conducted in a petroleum-contaminated aquifer to evaluate the potential for benzene, toluene, ethylbenzene, and xylenes (BTEX) biodegradation under enhanced nitrate-reducing conditions. Addition of nitrate resulted in loss of toluene, ethylbenzene, and m,p-xylenes (TEX) after an initial lag period of approximately 9 days. Losses of benzene were not observed over the 60-day monitoring period. Tracer breakthrough curves (BTCs) were analyzed to derive transport and biodegradation parameters, including advective velocities, retardation factors, dispersion coefficients, biodegradation rate constants, and nitrate utilization ratios. Using the parameters derived from the BTC analysis, numerical simulations of one of the tracer experiments were conducted using BIONAPL/3D [Molson, J., BIONAPL/3D User Guide, A 3D Coupled Flow and Multi-Component Reactive transport model. University of Waterloo, Waterloo, Ontario, Canada]. Simulations using the BTC-derived transport and biodegradation parameters successfully reproduced benzene, TEX, and nitrate concentrations measured during the tracer experiment. Comparisons of observed and simulated nitrate concentrations indicate that the mass ratio of nitrate-N utilized to TEX degraded increased over time during the experiment, reaching values many times that expected based on stoichiometry of TEX oxidation coupled to nitrate reduction. Excess nitrate loss is likely due to oxidation of other organics in addition to TEX.

Effects of dietary phytoestrogen on global myocardial ischemia-reperfusion injury in isolated female rat hearts.

We investigated the effects of phytoestrogen on global myocardial ischemia-reperfusion injury in five groups of female rats. A high-phytoestrogen group (HPE) was ovariectomized (Ovx) and fed a diet containing soybean protein and a high-isoflavone soy extract. Another Ovx group of rats was fed the same diet as the HPE group but treated with the estrogen receptor blocker ICI-182,780 (HPE + ICI). A third group of Ovx rats was fed a diet containing soybean protein alone (low-phytoestrogen content; LPE). A fourth Ovx group was fed a diet free of phytoestrogen (Ovx). The fifth group of rats was sham ovariectomized (sham). Hearts from all rats were subjected to 30 min of global, hypothermic (4 degrees C), cardioplegic ischemia and 120 min of normothermic (37 degrees C) reperfusion with oxygenated Krebs-Henseleit buffer. Compared with either the sham or the HPE group, the Ovx and HPE + ICI groups had significantly decreased first derivative of left ventricular pressure (dP/dt), coronary flow rate (CFR), nitrite production and mitochondrial respiratory function and significantly increased Ca2+ accumulation and myocardial histological and ultrastructural injury. The CFR of the LPE group was significantly different from that of either Ovx or HPE + ICI group but the dP/dt, nitrite production, Ca2+ accumulation, and mitochondrial function were not. Our results indicate that diets containing phytoestrogen extract play a cardioprotective role in global myocardial ischemia-reperfusion in female rats.

Chicken granulosa cells show differential expression of epidermal growth factor (EGF) and luteinizing hormone (LH) receptor messenger RNA and differential responsiveness to EGF and LH dependent upon location of granulosa cells to the germinal disc.

Granulosa cells in the chicken follicle exhibit different phenotypes according to their location relative to the germinal disc (GD). Granulosa cells proximal to the GD (referred to as proximal granulosa cells) are more proliferative, whereas granulosa cells distal to the GD (referred to as distal granulosa cells) are more differentiated. We have shown that epidermal growth factor (EGF) derived from the GD stimulated proliferation of granulosa cells proximal to the GD, whereas extraovarian LH promoted differentiation. We tested the hypothesis that phenotypic differences of granulosa cells are the result of differential responsiveness of granulosa cells to EGF and LH. We found that both granulosa and theca layers of chicken preovulatory follicles expressed mRNA for EGF receptor (EGFr) by polymerase chain reaction (PCR) analysis. However, only the granulosa layer showed differential expression of EGFr and LH receptor (LHr) mRNA. Competitive reverse transcription-PCR revealed that proximal granulosa cells expressed more EGFr mRNA but less LHr mRNA than distal granulosa cells. In addition, proximal granulosa cells proliferated more in response to EGF than their distal counterparts. We further demonstrated that EGF decreased LHr mRNA expression by granulosa cells in a dose-dependent manner, whereas EGF and LH had no effect on EGFr mRNA expression except at one dose of LH (15 ng/ml) that stimulated EGFr mRNA expression. Our findings suggest that EGF derived from the GD influences the phenotypes of granulosa cells. Granulosa cells proximal to the GD exhibit a proliferative phenotype possibly because they are exposed to and are more responsive to GD-derived EGF. Furthermore, GD-derived EGF decreases LHr mRNA expression by proximal granulosa cells and therefore results in less differentiated granulosa cell phenotype. In contrast, granulosa cells distal to the GD are not under the influence of EGF and exhibit a more differentiated phenotype.

Germinal disc-derived epidermal growth factor: a paracrine factor to stimulate proliferation of granulosa cells.

The germinal disc (GD) of the chicken oocyte produces factors that influence proliferation and differentiation of granulosa cells. Granulosa cells proximal to the GD are more proliferative, whereas granulosa cells distal to the GD are more differentiated. Previously, we had found epidermal growth factor (EGF) was present in the GD. In this study, we tested the hypothesis that EGF is the GD-derived paracrine factor that stimulates proliferation of granulosa cells. Northern analysis, reverse transcription-polymerase chain reaction, and radioimmunoassay indicated that the GD and granulosa cells but not theca cells are the sources of EGF in chicken preovulatory follicles. However, only the conditioned medium from the GD region (GDR = GD + overlying granulosa cells) but not the granulosa cell-conditioned medium stimulated proliferation of granulosa cells. Pretreatment of conditioned media with EGF antibody abolished the proliferation-stimulating effect of the GDR-conditioned medium. We conclude that EGF is one of the paracrine factors produced by the GD to stimulate proliferation of granulosa cells. Granulosa cells proximal to the GD express a proliferative phenotype possibly because they are exposed to a greater amount of EGF derived from the GD.

Estrogen receptor alpha has a functional role in the mouse rete testis and efferent ductules.

Previous studies of the estrogen receptor-alpha knockout (alpha ERKO) in the male mouse demonstrate that the rete testis and efferent ductules are targets of estrogen. Because the alpha ERKO mouse lacks a functional estrogen receptor alpha (ER alpha) throughout development, it was not known whether the morphological and physiological abnormalities observed in the alpha ERKO male were due to developmental defects or to dysfunctions concurrent with the lack of ER alpha in the tissue. This study was designed to determine if treatment of normal wild-type (WT) mice with the pure antiestrogen, ICI 182,780, (ICI) could reproduce the morphological characteristics seen in alpha ERKO mice. Thirty-day-old male mice were treated for 35 days with either castor oil or ICI. Age-equivalent alpha ERKO mice were used for comparison. Light microscopic examinations of the reproductive tracts revealed dramatic changes in the efferent ductules of treated mice: a 1.7-fold increase in luminal diameter, a 56% reduction in epithelial cell height, a 60% reduction in brush boarder height of nonciliated cells, and an apparent reduction of the number of observable lysosomes and endocytotic vesicles. Testes of ICI-treated mice showed swollen rete testes area (6.5 times larger than control) and a 65% reduction in rete testis epithelium height. However, there were no significant changes in body and testis weights. These results indicate that ER blockage with ICI in WT mice results in morphological changes of the efferent ductules resembling those seen in alpha ERKO siblings of the same age. Based on this study, we conclude that ER alpha has a functional role in the mouse reproductive tract and the aberrant morphology observed in the efferent ductules of the alpha ERKO mouse is likely the result of a concurrent response to the lack of functional ER alpha, and not solely due to the lack of ER alpha during early developmental times.

Effect of estrogen on global myocardial ischemia-reperfusion injury in female rats.

We investigated the effects of estrogen on global myocardial ischemia-reperfusion injury in rats that were ovariectomized (Ovx), sham-operated, or ovariectomized and then given 17beta-estradiol (E(2)beta) supplementation (Ovx+E(2)beta). Hearts were excised, cannulated, perfused with and then immersed in chilled (4 degrees C) cardioplegia solution for 30 min, and then retrogradely perfused with warm (37 degrees C), oxygenated Krebs-Henseleit bicarbonate buffer for 120 min. The coronary flow rate, first derivative of left ventricular pressure, and nitrite production were all significantly lower in Ovx than in sham-operated or Ovx+E(2)beta hearts. However, coronary flow rates or nitrate production were not consistently different throughout the entire reperfusion period. Ca(2+) accumulated more in Ovx rat hearts than in sham-operated or Ovx+E(2)beta hearts, and mitochondrial respiratory function was lower in Ovx hearts than in hearts from the other two groups. Marked interstitial edema and contraction bands were seen in hematoxylin-eosin-stained sections of Ovx rat hearts but not in hearts from either of the other groups. Hematoxylin-basic fuchsin-picric acid-stained sections revealed fewer viable myocytes in hearts from the Ovx group than from the sham or Ovx+E(2)beta group. Transmission electron microscopy demonstrated more severely damaged mitochondria and ultrastructural damage to myocytes in Ovx rat hearts. Our results indicate that estrogen plays a cardioprotective role in global myocardial ischemia-reperfusion injury in female rats.

Testicular recrudescence in the male black bear (Ursus americanus): changes in testicular luteinizing hormone-, follicle-stimulating hormone-, and prolactin-receptor ribonucleic acid abundance and dependency on prolactin.

Testicular recrudescence in male black bears (Ursus americanus) is initiated in January and completed in May. The goals of this study in the black bear were to determine 1) if testicular abundance of LH-receptor (LHr), FSH-receptor (FSHr), and prolactin-receptor (PRLr) mRNA changes during recrudescence; 2) if these changes in mRNA abundance are associated with changes in serum LH, PRL, and testosterone (T) concentrations; and 3) if the spring increase in serum PRL concentrations is required for testicular recrudescence. Serum was obtained monthly from nine male bears for 2 yr, except in July and August. To suppress endogenous PRL, four bears were treated with Parlodel LAR, 50 mg per 70 kg body weight, monthly from January through May, whereas five bears served as controls. Testicular biopsies were obtained in January, March, and May and analyzed for LHr, FSHr, and PRLr mRNA abundance using reverse transcriptase-competitive polymerase chain reaction. The LHr and PRLr mRNA abundance was low in January, increased in March, and remained high in May, whereas the FSHr mRNA abundance remained constant. Serum concentrations of PRL and T increased in March, coincident with the increase in testicular LHr and PRLr mRNA abundance. Suppression of serum PRL concentrations during testicular recrudescence 1) prevented the increase in testicular LHr and PRLr mRNA abundance observed among control bears in March, 2) lowered serum T concentrations in March and April, and 3) resulted in reduced testis size in May. We conclude that testicular LHr and PRLr mRNA are seasonally regulated, and that PRL has a role in testicular recrudescence in the black bear.

Identification of epididymal stones in diverse rooster populations.

The epididymal region of the male reproductive tract is essential for sperm maturation, and dysfunction of this region results in infertility. Adult roosters have been observed to develop epididymal stones and consequently have reduced fertility. Efferent ductule cysts were first observed in White Leghorn roosters ages 18 to 26 wk. By 26 wk of age, the cysts had become solid, irregularly shaped, yellow-green stones primarily containing calcium (48%). The number and size of stones (9 to 160 microm, largest diameter) increased with age in affected males. Incidence ranged from 0 to 94% within rooster flocks surveyed. Stones have also been observed in broiler breeder roosters. Histological analysis of Leghorn and broiler breeder reproductive tracts revealed chronic inflammation with abundant interstitial mononuclear cell infiltrates. The normal, highly folded structure of efferent ductules was replaced by a thin, eroded epithelial layer with few luminal sperm. Abnormal areas were found interspersed with normal areas of epithelium. Broiler breeder male fertility trials demonstrated that birds with stones compared with normal males had reduced fertility following both natural mating (24.8+/-10.5% vs. 66.1+/-7.2%) and artificial insemination (47.8+/-16% vs. 82.0+/-6%). At 62 wk of age, testis weight (14.2+/-1.4 g vs. 20.5+/-1.2 g), daily sperm production (8.1+/-1.3 x 10(8) vs. 12.3+/-0.8 x 10(8) sperm per testis per day), and circulating testosterone concentrations (0.9+/-0.3 vs. 2.6+/-0.4 ng/mL) were all significantly reduced in males with stones. In conclusion, we are reporting a new dysfunction of the rooster reproductive tract that affects diverse bird populations and decreases fertility.

Cloning and sequence analysis of the extracellular region of the polar bear (Ursus maritimus) luteinizing hormone receptor (LHr), follicle stimulating hormone receptor (FSHr), and prolactin receptor (PRLr) genes and their expression in the testis of the black bear (Ursus americanus).

Male black bears undergo seasonal changes in testicular activity. The testes are fully functional from May through July, regress from July through December, and recrudesce from January until May. The mechanisms responsible for the initiation of testicular recrudescence in the bear are unknown. The objectives of this study were to: (1) clone and sequence a substantial fragment of the extracellular portion of the luteinizing hormone receptor (LHr: 646 bp) and follicle stimulating hormone receptor (FSHr: 852 bp), and the extracellular/transmembrane portion of the prolactin receptor (PRLr: 680 bp) in the bear using reverse transcription-polymerase chain reaction (RT-PCR); and (2) determine whether the expression of LH-, FSH-, and PRL-receptor mRNA transcripts differs between the beginning and terminal stages of testicular recrudescence. Comparisons of the partial cDNA and predicted amino acid sequences of ursine receptors with the corresponding sequences from the pig, cow, human, and rat suggest that the LHr and FSHr are highly conserved (LHr: 87.1-93.7%; FSHr: 86.0-92.7%) whereas the PRLr is less well conserved (81-87%). Testicular LHr mRNA was more abundant during the breeding season in May than during the non-breeding season (early stage of recrudescence) in January. In contrast, testicular FSHr mRNA abundance was greater in January than in May. Testicular PRLr mRNA appeared equally abundant in January and May; however, two additional transcripts were present during the breeding season in May. This study provides molecular tools for future investigations of the control of testicular recrudescence in the black bear and demonstrates that the expression of testicular gonadotropin and PRL receptor mRNA is seasonally regulated. Mol. Reprod. Dev. 55:136-145, 2000.

Molecular analysis of microbial community structures in pristine and contaminated aquifers: field and laboratory microcosm experiments.

This study used phylogenetic probes in hybridization analysis to (i) determine in situ microbial community structures in regions of a shallow sand aquifer that were oxygen depleted and fuel contaminated (FC) or aerobic and noncontaminated (NC) and (ii) examine alterations in microbial community structures resulting from exposure to toluene and/or electron acceptor supplementation (nitrate). The latter objective was addressed by using the NC and FC aquifer materials for anaerobic microcosm studies in which phylogenetic probe analysis was complemented by microbial activity assays. Domain probe analysis of the aquifer samples showed that the communities were predominantly Bacteria; Eucarya and Archaea were not detectable. At the phylum and subclass levels, the FC and NC aquifer material had similar relative abundance distributions of 43 to 65% beta- and gamma-Proteobacteria (B+G), 31 to 35% alpha-Proteobacteria (ALF), 15 to 18% sulfate-reducing bacteria, and 5 to 10% high G+C gram positive bacteria. Compared to that of the NC region, the community structure of the FC material differed mainly in an increased abundance of B+G relative to that of ALF. The microcosm communities were like those of the field samples in that they were predominantly Bacteria (83 to 101%) and lacked detectable Archaea but differed in that a small fraction (2 to 8%) of Eucarya was detected regardless of the treatment applied. The latter result was hypothesized to reflect enrichment of anaerobic protozoa. Addition of nitrate and/or toluene stimulated microbial activity in the microcosms, but only supplementation of toluene alone significantly altered community structures. For the NC material, the dominant subclass shifted from B+G to ALF, while in the FC microcosms 55 to 65% of the Bacteria community was no longer identifiable by the phylum or subclass probes used. The latter result suggested that toluene exposure fostered the proliferation of phylotype(s) that were otherwise minor constituents of the FC aquifer community. These studies demonstrated that alterations in aquifer microbial communities resulting from specific anthropogenic perturbances can be inferred from microcosm studies integrating chemical and phylogenetic probe analysis and in the case of hydrocarbon contamination may facilitate the identification of organisms important for in situ biodegradation processes. Further work integrating and coordinating microcosm and field experiments is needed to explore how differences in scale, substrate complexity, and other hydrogeological conditions may affect patterns observed in these systems.

Sex steroid and prolactin profiles in male American black bears (Ursus americanus) during denning.

Serum sex steroid and prolactin profiles were examined in the male American black bear, Ursus americanus during denning. Sera collected in December and the following March from 8 denning male black bears in Minnesota, U.S.A. were assayed for testosterone, estradiol-17 beta and prolactin. Eight bears were confirmed to be the denning mode based on a serum urea to creatinine ratio less than 10. Serum testosterone concentrations tended to increase from December to the subsequent March whereas serum estradiol-17 beta concentrations tended to decrease during this period. There were few changes in serum prolactin concentrations between December and March. These findings suggest that spermatogenesis and testicular steroidogenesis initiated during denning may be influenced by changes in serum sex steroid concentrations in the American black bear.

Restricted expression of WT1 messenger ribonucleic acid in immature ovarian follicles: uniformity in mammalian and avian species and maintenance during reproductive senescence.

WT1 is a zinc finger protein with transcriptional repressor activity on several growth factor and growth factor receptor genes. In the ovary, a potential role for WT1 in the suppression of the development of immature follicles has been demonstrated. Here, gel retardation assays further showed that recombinant WT1 protein interacted with consensus DNA sequences in the inhibin-alpha gene promoter. We investigated the pattern of WT1 expression in a wide variety of species and also over the reproductive life span in rats. In chicken ovaries, Northern blot analysis revealed the presence of WT1 transcript in small healthy white follicles (1-5 mm in diameter) and its absence in small yellow (6-12 mm in diameter) or larger follicles (F1-F5). In pig and monkey ovaries, WT1 expression was limited to granulosa cells of preantral follicles, as shown by in situ hybridization analysis. In rats, Northern blot analyses demonstrated the presence of WT1 transcript in the ovaries of young (3-mo-old) and middle-aged (9-mo-old) rats on the proestrous day, with a decrease in old (12-mo-old) rats in persistent estrus. In situ hybridization analysis further suggested that the decrease in WT1 expression in aging ovaries was associated with fewer immature follicles. Thus, WT1 expression is restricted to immature follicles in diverse avian and mammalian species and over the reproductive life span in rats. These data demonstrated that WT1 is a marker for immature follicles and suggested a potential role of this transcriptional repressor in the slow growth of early follicles.