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Mosquitofish (Gambusia) are sexually dimorphic; adult males have an elongation of the anal fin to form a copulatory structure known as a gonopodium. Several studies since the early 1980s have reported elongated anal fins in female mosquitofish exposed to pulp and paper mill discharge, a phenomenon known as masculinization. Although adverse impacts have been suggested in these previous reports, the influence of masculinization on reproductive success has not been assessed for pulp mill effluent-exposed eastern mosquitofish (Gambusia holbrooki or affinis). The current study compared fecundity (number of fry per female at parturition) to an index of masculinization (ratio of anal fin rays 4 to 6). Pregnant females were collected from two effluent-receiving streams in Florida: Rice Creek and the Fenholloway River, over two reproductive seasons (2003 and 2004). Masculinization was consistent between years, with clear effects at the Fenholloway River site, while the response was minimal or nondetectable at the Rice Creek site. Masculinization was not correlated with the production of fry/fecundity at either site. Data suggest differing reproductive seasonal strategies between basins and populations but do not demonstrate any effects of pulp and paper exposure on reproductive success in mosquitofish.

The abnormal induction of anal fin elongation (masculinization) in female Gambusia was first reported in the 1980s for Florida streams receiving pulp and paper effluents. Although these early reports indicated masculinization responses that were similar to the complete development of a gonopodium (male secondary sex structure), additional evaluations throughout the 1990s demonstrated significant reductions in this response. These historic data suggested that mill process modifications may have been responsible for the reduced masculinization responses. The objective of the current study was to utilize Georgia Pacifics Palatka Mill to monitor a series of mill process modifications and their effects on masculinization responses in Mosquitofish (Gambusia). Gambusia were collected from upstream, discharge, and downstream sites in the Palatka Mill receiving stream (Rice Creek) and masculinization was evaluated. Collections were conducted annually during 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2008, and 2009, before and after a series of modifications which included: conversion to elemental chlorine free bleaching, conversion of secondary treatment to aerobic degradation, reduction of in-mill black liquor losses, addition of condensate stripping, reduction in water usage from 136 to 83 million litres per day (36 to 22 million gallons per day), installation of new brown stock washers, and oxygen delignification. Masculinization responses were evaluated using the anal fin index (ratio or anal fin rays 4 and 6). Reductions in this masculinization response were observed gradually across 1999 through 2009, with an absence of response in female Gambusia during 2004, 2006, and 2008 to 2009. These data indicate that pulp and paper mill upgrades and process modifications have resulted in the elimination of this biological response in Gambusia.

In arid regions of the southwestern United States, municipal wastewater treatment plants commonly discharge treated effluent directly into streams that would otherwise be dry most of the year. A better understanding is needed of how effluent-dependent waters (EDWs) differ from more natural aquatic ecosystems and the ecological effect of low levels of environmentally persistent organic wastewater compounds (OWCs) with distance from the pollutant source. In a controlled experiment, we found 26 compounds common to municipal effluent in treatment raceways all at concentrations1.0 microg/L. Male bonytail chub (Gila elegans) in tanks containing municipal effluent had significantly lower levels of 11-ketotestosterone (p=0.021) yet higher levels of 17beta-estradiol (p=0.002) and vitellogenin (p=0.036) compared to control male fish. Female bonytail chub in treatment tanks had significantly lower concentrations of 17beta-estradiol than control females (p=0.001). The normally inverse relationship between primary male and female sex hormones, expected in un-impaired fish, was greatly decreased in treatment (r=0.00) versus control (r=-0.66) female fish. We found a similar, but not as significant, trend between treatment (r=-0.45) and control (r=-0.82) male fish. Measures of fish condition showed no significant differences between male or female fish housed in effluent or clean water. Inter-sex condition did not occur and testicular and ovarian cells appeared normal for the respective developmental stage and we observed no morphological alteration in fish. The population-level impacts of these findings are uncertain. Studies examining the long-term, generational and behavioral effects to aquatic organisms chronically exposed to low levels of OWC mixtures are needed.

In this study, largemouth bass (LMB) were subchronically exposed to p,p'-DDE or dieldrin in their diet to evaluate the effect of exposure on expression of genes involved in reproduction and steroid homeostasis. Using real-time PCR, we detected a different gene expression pattern for each OCP, suggesting that they each affect LMB in a different way. We also detected a different expression pattern among sexes, suggesting that sexes are affected differently by OCPs perhaps reflecting the different adaptive responses of each sex to dysregulation caused by OCP exposure.

The objective of this study was to evaluate the effects of bleached/unbleached kraft mill effluents (B/UKME) on the reproductive parameters of free-ranging Florida largemouth bass (Micropterus salmoides floridanus). The reproductive parameters measured included gonadosomatic index (GSI), histological evaluation of gonads, and plasma concentrations of vitellogenin (VTG), 17beta-estradiol, and 11-ketotestosterone (11-KT). Hepatic ethoxyresorufin-O-deethylase (EROD) activity was measured as a marker of exposure to cytochrome P450-inducing agents in these effluents. Endpoints were compared among adult bass sampled from tributary and mainstream effluent-contaminated and reference sites. Females sampled from the site closest to the mill outfall had a significant five-fold increase in EROD activity compared to bass sampled from reference streams. Although sex hormones were significantly reduced in bass from exposed sites, there were no differences in VTG and GSI across sites. The absence of organism-level responses was probably not related to a lack of sensitivity, as previous studies in our laboratory have shown that bass exposed to these effluents exhibit changes in GSI and in other measures associated with reproductive success. In females, inverse relationships were observed between VTG and GSI and EROD activity. These relationship, however, were not consistent within all of the sites studied. Collectively, our findings indicate that hepatic EROD induction is an effective marker of B/UKME exposure in largemouth bass and that it might be associated with antiestrogenic effects in this species.

In the 1980s, alligators (Alligator mississippiensis) of Lake Apopka (Florida, USA) underwent a population decline associated with decreased egg viability, effects that have been associated with endocrine-disrupting, persistent organochlorine pesticides. It is currently unknown whether the decreased egg viability is due to fertilization failure or early embryonic death. Therefore, we conducted a preliminary study to evaluate the use of microsatellite DNA loci to determine the fertilization status of nonviable eggs. Using microsatellite analysis, we compared genotypes from blasto-disks and embryos with the genotypes from females trapped at the nest. Four of five nonviable egg samples tested yielded evidence of fertilization. No evidence of unfertilized eggs was obtained, but amplifiable DNA could not be obtained from one entirely nonviable clutch. Thus, we demonstrate that early embryonic mortality in alligators can be detected by microsatellite analysis, but also suggest substantial effort is needed to improve the recovery of DNA and amplification of alligator microsatellite loci.

This study reports baseline adrenocortical, hematological, and plasma biochemical values for clinically healthy juvenile green turtles from a discrete population at Kaneohe Bay, island of Oahu, Hawaii. Using a general linear modeling program, we compared mean values for these parameters with mean values of a group afflicted with green turtle fibropapillomas (GTFP). Turtles of similar size classes from both groups were collected under the same conditions in the same study area and season at the same time of the day. Corticosterone, hematological, and enzymatic responses to acute and chronic stress were characterized for each group at four different sampling periods: 0 h (within 2 min of capture), 1 h, 3-4 h, and 24 h postcapture. On the basis of the differences identified between groups and times within a group, we conclude that turtles with GTFP are chronically stressed and immunosuppressed.

The reproductive development of alligators from a contaminated and a control lake in central Florida was examined. Lake Apopka is adjacent to an EPA Superfund site, listed due to an extensive spill of dicofol and DDT or its metabolites. These compounds can act as estrogens. Contaminants in the lake also have been derived from extensive agricultural activities around the lake that continue today and a sewage treatment facility associated with the city of Winter Garden, Florida. We examined the hypothesis that an estrogenic contaminant has caused the current failure in recruitment of alligators on Lake Apopka. Supporting data include the following: At 6 months of age, female alligators from Lake Apopka had plasma estradiol-17 beta concentrations almost two times greater than normal females from the control lake, Lake Woodruff. The Apopka females exhibited abnormal ovarian morphology with large numbers of polyovular follicles and polynuclear oocytes. Male juvenile alligators had significantly depressed plasma testosterone concentrations comparable to levels observed in normal Lake Woodruff females but more than three times lower than normal Lake Woodruff males. Additionally, males from Lake Apopka had poorly organized testes and abnormally small phalli. The differences between lakes and sexes in plasma hormone concentrations of juvenile alligators remain even after stimulation with luteinizing hormone. Our data suggest that the gonads of juveniles from Lake Apopka have been permanently modified in ovo, so that normal steroidogenesis is not possible, and thus normal sexual maturation is unlikely. Images p680-a Figure 1. Figure 2. Figure 3. A Figure 3. B Figure 3. C Figure 4. A Figure 4. B Figure 4. C Figure 4. D Figure 5. A Figure 5. B Figure 5. C

Plasma concentrations of estradiol, progesterone, prostaglandin (PG) E2 and PGF were measured during three stages of the natural nesting cycle of the tuatara (Sphenodon punctatus), a rare New Zealand reptile. Specifically, we tested whether nesting activity in this species is correlated with significant modifications in plasma concentrations of these hormones. Tuatara dig a nest over a period of several nights, oviposit a complete clutch on a single night, and then guard that nest for several nights. We observed no significant changes in mean plasma concentrations of estradiol or progesterone among the different stages of nesting. In contrast, PGE2 was elevated during nest digging and oviposition but declined significantly during nest guarding. Plasma PGF was elevated during nest digging, rose significantly during oviposition, and declined to basal levels during nest guarding. These data indicate that oviposition in reptiles is associated with elevated plasma concentrations of PGs, suggesting that these hormones function in causing oviducal contractions and/or luteolysis. Moreover, these data suggest that the role of PGs in oviducal function may have been conserved throughout evolution of the amniote vertebrates.

Reproductive success and development of F2 offspring from F1 adult African clawed frogs (Xenopus laevis) exposed to atrazine throughout larval development and as sexually mature adults was examined. Larval X. laevis were exposed to one of four nominal concentrations of atrazine (0, 1, 10, 25 microg atrazine/l) beginning 96 hr after fertilization and continuing through two years post-metamorphosis. Clutch size and survival of offspring were used as measurement endpoints to gauge reproductive success of the F1 frogs. Larval survivorship and time to metamorphosis were used to gauge developmental success of the F2 offspring from atrazine-exposed frogs. Testes in F1 and F2 frogs were examined for incidence of anomalies, such as testicular ovarian follicles, and sex ratios in F2 offspring were investigated to determine if exposure to atrazine caused trans-generational effects (effects on F2 individuals due to exposure of F1 individuals). There were no effects of any of the studied concentrations of atrazine on clutch size of F1 frogs. There were also no effects on hatching success or time to metamorphosis. Sex ratios did not differ between F2 offspring among treatments. There was no evidence to suggest a transgenerational effect of atrazine on spawning success or reproductive development of X. laevis. This is consistent with the presence of robust populations of X. laevis in areas where they are exposed to atrazine that has been used for several decades for weed control in production of corn. Our observations also are consistent with the results of most other studies of frogs where no effects were found to be associated with exposure to atrazine. Our data do not support the hypothesis that atrazine significantly affects reproductive fitness and development of frogs.

Dieldrin and p,p'-DDE are ubiquitous contaminants known to act as endocrine disruptors, causing impaired development and reproduction in fish and wildlife. In order to elucidate the mechanisms by which dieldrin and p,p'-DDE cause endocrine disruption in largemouth bass (Micropterus salmoides), fish were exposed subchronically through the diet to both contaminants. Following 120 days of exposure, p,p'-DDE decreased estradiol in females, but increased 11-ketotestosterone in both sexes. Dieldrin on the other hand, decreased estradiol and 11-ketotestosterone in both sexes. Both pesticides also altered steady state mRNA expression levels of a set of genes chosen to represent three possible mechanisms of endocrine disruption: (1) direct interaction with soluble sex steroid receptors, (2) biosynthesis of endogenous sex hormones, and (3) metabolism of endogenous hormones. p,p'-DDE acted as a weak estrogen, increasing the expression of vitellogenin and estrogen receptor alpha in the liver. p,p'-DDE also altered the expression of genes involved in the synthesis of endogenous hormones as well as their metabolism. Dieldrin, on the other hand, only altered expression of vitellogenin and not estrogen receptor alpha. Dieldrin also altered the expression of genes involved in hormone synthesis and metabolism, and it dramatically lowered plasma hormone levels. Both pesticides targeted expression of genes involved in all three modes of action, suggesting that they each have multiple modes of action.

The objective of this study was to characterize concentrations of atrazine, terbuthylazine, and other pesticides in amphibian habitats in surface waters of a corn-production area of the western Highveld region (North-West Province) of South Africa. The study was conducted from November 2001 to June 2002, coinciding with the corn-production season. Pesticide residues were measured at regular intervals in surface water from eight ponds, three in a non-corn-growing area (NCGA) and five within the corn-growing area (CGA). Measured atrazine concentrations differed significantly among sites and between samples. In the five CGA sites, the maximum atrazine concentrations measured during the study ranged from 1.2 to 9.3 mg/L. Although no atrazine was recorded as being applied in the catchment of the three NCGA sites, maximum concentrations from 0.39 to 0.84 mg/L were measured during the study, possibly as a result of atmospheric transport. Maximum measured concentrations of terbuthylazine ranged from 1.22 to 2.1 mg/L in the NCGA sites and from 1.04 to 4.1 mg/L in the CGA sites. The source of terbuthylazine in the NCGA sites may have been in use other than in corn. The triazine degradation products, deisopropylatrazine (DIA) and deethylatrazine (DEA) and diaminochlorotriazine (DACT) were also found in water from both the CGA and NCGA sites. Concentrations of DIA were R 1 mg/L throughout the season, while DEA concentrations were mostly !0.5 mg/L before planting but increased after planting and application of herbicides to concentrations O2 mg/L in some locations. Concentrations of DACT were highly variable (LOD to 8 mg/L) both before and after planting and application, suggesting that they resulted from historical use of triazines in the area. Other herbicides such as simazine and acetochlor were only detected infrequently and pesticides such as S-metolachlor, cypermethrin, monocrotophos, and terbuphos, known to be used in the CGA, were not detected in any of the samples. Because of dilution by higher than normal rainfall in the study period, these concentrations may not be predictive of those in years of normal rainfall. 2004 Elsevier Ltd. All rights reserved.

A number of freshwater lakes and reclaimed agricultural sites in Central Florida have been the receiving waters for agrochemical and municipal runoff. One of these sites, Lake Apopka, is also a eutrophic system that has been the focus of several case studies reporting altered reproductive activity linked to bioaccumulation of persistent organochlorine chemicals in aquatic species. The present study was initiated to determine if brown bullheads ( Ameiurus nebulosus ) from the north marsh of Lake Apopka (Lake Apopka Marsh) exhibit an altered capacity to detoxify environmental chemicals through hepatic glutathione S -transferase (GST)-mediated conjugation as compared with bullheads from a nearby reference site (Lake Woodruff). We also compared plasma sex hormone concentrations (testosterone, 17-β estradiol, and 11 keto-testosterone) in bullheads from the two sites. Female bullheads from Lake Apopka had 40% lower initial rate GST conjugative activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 50% lower activity towards p -nitrobutyl chloride (NBC), 33% lower activity toward ethacrynic acid (ECA), and 43% lower activity toward Δ5-androstene-3,17-dione (Δ 5 -ADI), as compared with female bullheads from Lake Woodruff. Enzyme kinetic analyses demonstrated that female bullheads from Lake Apopka had lower GST-catalyzed CDNB clearance than did female Lake Woodruff bullheads. Western blotting studies of bullhead liver cytosolic proteins demonstrated that the reduced GST catalytic activities in female Lake Apopka bullheads were accompanied by lower expression of hepatic GST protein. No site differences were observed with respect to GST activities or GST protein expression in male bullheads. Female Lake Apopka bullheads also had elevated concentrations of plasma androgens (testosterone and 11-ketotestosterone) as compared with females from Lake Woodruff. In contrast, male Lake Apopka bullheads had elevated levels of plasma estrogen but similar levels of androgens as compared with male bullheads from Lake Woodruff. Collectively, our studies indicate the presence of reduced GST protein expression, reduced GST conjugative capacity and altered sex steroid homeostasis in female bullheads from a contaminated field site in Central Florida. The implications of these physiological alterations in terms of pollutant biotransformation and reproduction are discussed.

This document describes procedures used by biologists to collect information, tissues, and fluids useful for determining the exposure of fish to environmental contaminants. Fish are to be captured and held alive (generally

A reconnaissance of sex steroid hormones and other biomarkers in common carp was used to assess whether endocrine disruption may be occurring in fish in United States streams, to evaluate relations between endocrine disruption and contaminant levels, and to determine requirements for further studies. 17s-estradiol, 11-ketotestosterone, vitellogenin, and gonadal histopathology were measured in adult carp (usually 10--15 for each sex) at 25 sites (647 fish), representing a wide range of environmental settings typical of major regions of the nation. Fish were collected during August--December 1994, a period of gonadal maturation after spawning. Contaminants evaluated were organochlorine pesticides and polychlorinated biphenyls in tissue; phthalates, phenols, and polycyclic aromatic hydrocarbons in bed sediment; and dissolved pesticides in water. Mean site concentrations of steroid hormones spanned two orders of magnitude for both sexes. No significant regional differences in steroid hormones were detected for males, but females from the Northern and Southern Midcontinent were significantly different from other regions of the country in one or both hormones. Within all regions there were significant differences between sites in one or both hormones for both sexes. Most correlation coefficients between biomarkers and contaminants were negative. Contaminants that had significant (a=0.05) correlations with biomarkers were organochlorine pesticides, phenols, and dissolved pesticides. The strongest pattern common to both males and females was a negative correlation between the hormone ratio (E2/11-KT) and dissolved pesticides. The significant site-to-site differences in biomarkers, and the presence of significant correlations between biomarkers and contaminants, are evidence that fish in some streams may be experiencing endocrine disruption. Improved information is needed to evaluate whether endocrine disruption is actually occurring and if there are reproductive effects on individual or populations of carp or other species. Future studies should shift to more intensive study of fewer sites, including reference and contaminated sites, in order to address these additional questions.

Developing assays to detect endocrine-mediated toxicity from in ovo or in utero exposure is a current challenge in regulatory toxicology. Some species of reptiles exhibiting a unique mode of sex determination, in which the incubation temperature during a critical period determines gonadal sex, have been explored as an in ovo model to screen environmental contaminants for endocrine effects. We critically review published egg-exposure studies and conclude that data regarding the pharmacokinetics of topically applied substances are insufficient to validate dose-response relationships for the effects of chemicals on in ovo endocrine function or gender determination in reptiles. The insufficiencies in these data largely result from methodological failures, including lack of measurement verification, failure to investigate and control extraneous factors affecting the measurements, and lack of independent replication of results. Considerable additional research will be necessary to alleviate these methodological inadequacies. Given the current status of the data, topical treatment of reptilian eggs cannot be considered to be a valid means of establishing causal relationships between chemical treatment and biological outcome.

The ubiquitous distribution of many contaminants and the nonlethal, multigenerational effects of such contaminants on reproductive, endocrine, and immune systems have led to concerns that wildlife worldwide are affected. Although the causal agents and effects are known for some species, the underlying physiological mechanisms associated with contaminant-induced reproductive modifications are still poorly understood and require extensive research. We describe a study examining the steroidogenic activity of gonads removed from juvenile alligators (Alligator mississippiensis) obtained from contaminated or control lakes in central Florida. Synthesis of estradiol-17 beta (E2) was significantly different when ovaries from the contaminated and control lakes were compared in vitro. Additionally, testes from males obtained from the contaminated lake. Lake Apopka, synthesized significantly higher concentrations of E2 when compared to testes obtained from control males. In contrast, testosterone (T) synthesis from all testes examined in this study displayed a normal pattern and produced concentrations greater than that observed from ovaries obtained from either lake. Interestingly, the pattern of gonadal steroidogenesis differs from previously reported plasma concentrations of these hormones obtained from the same individuals. We suggest that the differences between the in vivo and in vitro patterns are due to modifications in the hepatic degradation of plasma sex steroid hormones.

Many of the remaining members of the endangered Florida panther (Felis concolor coryi) population suffer from one or more of a variety of physiological, reproductive, endocrine, and immune system defects including congenital heart defects, abnormal sperm, low sperm density, cryptorchidism, thyroid dysfunction, and possible immunosuppression. Mercury contamination, determined to be the cause of death of a female panther in 1989, was presented as the likely cause of thyroid dysfunction. As genetic diversity in the species was less than expected, all of the other abnormalities have been attributed to inbreeding. However, exposure to a variety of chemical compounds, especially those that have been identified as environmental endocrine disrupters (including mercury, p,p'-DDE, and polychlorinated biphenyls), has elicited all of the listed abnormalities in other species. A number of these contaminants are present in South Florida. An exposure pathway has been identified, and evidence presented in this paper, including the fact that there appears to be no significant difference between serum estradiol levels in males and females, suggests that many male panthers may have been demasculinized and feminized as a result of either prenatal or postnatal exposure. Thus, regardless of the effects of inbreeding, current evidence seems to indicate that environmental contaminants may be a major factor contributing to reproductive impairment in the Florida panther population.

The conceptus is believed to produce factors that regulate endometrial function and prevent luteolysis during early pregnancy. Endometrial tissues were collected from cyclic (n = 8) and pregnant (n = 2) cows at Day 17 post-estrus and cultured for 24 and 48 h with bovine conceptus secretory proteins (bCSP) (0%, 10%, 100%), where the amount of protein produced by a bovine conceptus during 24 h of culture is 100%. Incorporation of [3H]leucine into secreted proteins was determined and examined qualitatively by trichloroacetic acid precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Levels of an intracellular endometrial inhibitor of prostaglandin synthesis were determined with a cotyledonary microsomal test system. Treatment with 10% and 100% bCSP reduced incorporation of [3H]leucine into secreted proteins. However, bCSP selectively induced two secreted proteins (13 and 10 kDa) from endometrium of cyclic cows. Prostaglandin F (PGF) secretion was decreased by bCSP treatment while prostaglandin E2 secretion was unaltered. An intracellular endometrial inhibitor of prostaglandin synthesis was induced by bCSP; synthesis of PGF by the cotyledonary prostaglandin-generating system was decreased when incubated with cytosol of endometrium treated with bCSP, but unaltered by cytosol from control tissues. In conclusion, products produced by the bovine conceptus are capable of regulating endometrial protein and prostaglandin biosynthesis in a fashion that could act to prevent luteolysis in vivo and provide endometrial secretory products for embryonic development.

Endometrium from cyclic (N = 4) and pregnant (N = 4) gilts at Day 14 after oestrus was placed into three bilateral perifusion devices which allow separate perifusion of luminal and myometrial sides. Perifused endometrium was subjected to 39 or 42 degrees C. Incorporation of [3H]leucine into secreted and tissue proteins by endometrial explants following incubation at 39 or 42 degrees C was examined using trichloroacetic acid (TCA) precipitation and one-dimensional SDS polyacrylamide gel electrophoresis. Secretion of PGF was greater from the myometrial side for cyclic gilts (endocrine orientation), but greater from the luminal side for pregnant gilts (exocrine orientation). Regardless of reproductive status or endometrial side, heat stress induced a rapid increase (P less than 0.01) in PGF secretion rates. However, PGF secretion in response to heat stress was greater (P less than 0.01) from the myometrial side and greater (P less than 0.01) for pregnant gilts. PGF secretion rates increased by 63% and 42% from the luminal side, and 40% and 156% from the myometrial side in response to heat stress for cyclic and pregnant gilts, respectively (status x treatment x side interaction; P less than 0.01). Heat stress did not alter incorporation of [3H]leucine into secreted proteins regardless of reproductive status, while incorporation into tissue proteins was decreased (P less than 0.05) by heat stress for pregnant gilts, but not altered for cyclic gilts. Heat stress, in vitro, redirects PGF secretion for endometria of pregnant gilts from an exocrine to an endocrine orientation where it would be available to effect luteolysis and compromise the establishment of pregnancy.

Lactating pregnant and nonpregnant dairy cows were used to determine if summer heat stress alters responsiveness of the uterus to an oxytocin-induced secretion of prostaglandin F 2α . Cows were assigned to thermoneutral (evaporatively-cooled barn), or heat stress (exposure to summer environment) treatments for 7.5 h on Day 17 post-estrus. Blood samples were collected during the first 6 h. Oxytocin (100 IU) was then administered and blood collected for a further 1.5 h. Conceptuses were recovered nonsurgically to determine pregnancy status. Prior to oxytocin treatment, plasma concentrations of 13,14-dihydro-15-keto PGFM 2α (PGFM) were similar among cows, regardless of treatment or pregnancy status. Oxytocin increased mean PGFM concentrations, but the increase was higher for nonpregnant and pregnant cows with embryos retarded in development than for pregnant cows with normal embryos. Heat stress increased oxytocin-induced PGFM concentrations in nonpregnant cows, but had no effect on PGFM in pregnant cows. Endometrial explants from cyclic and pregnant cows were used to determine if heat-induced endometrial secretion of prostaglandin (PG, PGF and PGE 2 ) is regulated by an intracellular inhibitor of PG synthesis. Endometrial explants were incubated at 39 or 42°C, and conditioned medium analyzed for PGF and PGE 2 . Cotyledonary microsomes from parturient cows were utilized as a PG generating system for detection of endometrial inhibitors of PG synthesis. Endometrial synthesis of PGE at 39°C was greater for cyclic than pregnant cows, while PGE 2 synthesis was similar between statuses. Heat stress increased PGF and PGE 2 synthesis by endometrium from cyclic and pregnant cows. Synthesis of PGF by the generating system was decreased 44% when incubated with endometrium of pregnant cows cultured at 39°C and this decrease was 35.3% less with cytosol from endometrium of pregnant cows incubated at 42°C. Decreased uterine PGF production by pregnant cows, carrying viable embryos, in response to oxytocin in vivo and by endometrial explants from pregnant cows, indicates that the conceptus attenuates release of uterine PGF. Heat stress amplified PG synthesis by nonpregnant cows and in endometrial explants from cyclic and pregnant cows. Decreased responsiveness of the pregnant uterus to oxytocin and heat stress suggests a conceptus-induced factor attenuates responsiveness of the uterine PG generating system to stimulation by heat stress.

Bilateral perifusion devices were utilized to measure prostaglandin F-2 alpha (PGF) secretion by bovine endometrium in response to in-vitro heat stress. Tissues were collected at Day 17 after oestrus from cyclic (N = 4) and pregnant (N = 5) cows, placed into 3 perifusion devices, perifused (3 ml/10 min, Krebs-Ringer-bicarbonate [KRB]) for 5 h, and fractions were collected every 10 min. Endometrial tissues within each device were subjected to a different temperature and oxytocin (1 i.u./ml KRB) treatment sequence: (1) control-oxytocin: 1 h at 39 degrees C; 2 h at 39 degrees C, 0.5 h at 39 degrees C with oxytocin, 0.5 h at 39 degrees C and 1 h at 39 degrees C; (2) heat-oxytocin: 1 h at 39 degrees C, 2 h at 42 degrees C, 0.5 h at 42 degrees C with oxytocin, 0.5 h at 42 degrees C and 1 h at 39 degrees C; (3) heat-KRB: 1 h at 39 degrees C, 2 h at 42 degrees C, 0.5 h at 42 degrees C, 0.5 h at 42 degrees C and 1 h at 39 degrees C. Regardless of reproductive status, heat stress induced a rapid increase (P less than 0.01) in PGF secretion rates. Oxytocin induced an increase (P less than 0.01) in PGF secretion for endometrium from cyclic cows regardless of temperature. Endometria from pregnant cows did not respond to oxytocin when perifused at 39 degrees C. However, PGF secretion rates from endometrium of pregnant cows increased (P less than 0.01) in response to oxytocin when perifused under heat stress conditions.(ABSTRACT TRUNCATED AT 250 WORDS)