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The onset of fear-related avoidance behavior occurs during and, to some extent, defines the sensitive period of development in the domestic dog. The objectives of this study were to identify the onset of fear-related avoidance behavior and examine breed differences in this behavioral development. A total of 98 purebred puppies representing 3 breeds were tested, namely Cavalier King Charles spaniels (n = 33), Yorkshire terriers (n = 32), and German shepherd dogs (n = 33). Data were collected weekly beginning 4-5 weeks after birth until 10 weeks of age. Puppies took part in 4 tests during each visit: a novel item, seesaw, step, and loud noise test. During each test, the presence or absence of fear-related avoidance behavior and crouched posture were noted. Saliva was also collected to measure salivary cortisol concentrations in the puppies before and after testing. A later onset of fear-related avoidance behavior was observed in Cavalier King Charles spaniels compared with German shepherd dog and Yorkshire terrier puppies (F = 11.78, N = 29, P

Contents The corpus luteum (CL) is an ephemeral endocrine organ. During its lifespan, it undergoes a period of extremely rapid growth that involves hypertrophy, proliferation and differentiation of the steroidogenic cells, as well as extensive angiogenesis. The growth phase is followed by a period in which remodelling of the tissue ceases, but it engages in unparalleled production of steroids, resulting in extraordinarily high metabolic activity within the tissue. It is during this stage that a critical juncture occurs. In the non-fertile cycle, uterine release of prostaglandin (PG)F2α initiates a cascade of events that result in rapid loss of steroidogenesis and destruction of the luteal tissue. Alternatively, if a viable embryo is present, signals are produced that result in rescue of the CL. This review article summarizes the major concepts related to the fate of the CL, with particular focus on recent insights into the mechanisms associated with the ability of PGF2α to bring about complete luteolysis. It has become clear that the achievement of luteolysis depends on repeated exposure to PGF2α and involves coordinated actions of heterogeneous cell types within the CL. Together, these components of the process bring about not only the loss in progesterone production, but also the rapid demise of the structure itself.

We systematically investigated the feminization of bluegill Lepomis macrochirus by oral administration of various doses of estradiol-17β (E 2 ) and evaluated their effects on the growth performance, production and gonadal structure of sex-reversed female bluegill at both sex ratio and histological levels. With positive control treatment, 30-day-old fry were fed with E 2 at 50, 100, 150 and 200 mg kg − 1 diet for 60 days. The survival of fish in the E 2 treated and control groups was not significantly different ( P > 0.05). The growth of the treated fish was significantly retarded during the period of treatment, while there was no side effect detected post-treatment and the retarded fish caught up during 120 days of culture after E 2 treatment. All the treated groups produced 100% monosex female populations based on the macroscopic shape of gonads, and there were no significant differences detected between any E 2 treatment and control group in the mean GSI of females during the spawning season from June to October ( P > 0.05). Histologically, 13.3% and 5.0% of the intersex fish were determined to come from the 50 and 100 mg kg − 1 E 2 treatment groups, respectively, with 6.9% and 4.1% of the gonadal area containing spermatocytes. Most of genotypical male fish treated with exogenous E 2 developed gonadal structures histologically indistinguishable from the gonads of females. This study suggests that 150 mg kg − 1 E 2 is the optimal dosage for feminization in bluegill, with 50 and 100 mg kg − 1 E 2 being sub-optimal and 200 mg kg − 1 E 2 being over-optimal.

A long-term feeding experiment (35 months) was conducted to study the effects of substitution of fish meal (FM) by cottonseed meal (CM) on growth, feed utilization and reproductive physiology in rainbow trout ( Oncorhynchus mykiss ). Five groups of fish (1.5 years old; 246 ± 10 g) were fed one of five experimental diets formulated to substitute FM with CM (147, 294, 441 and 588 g/kg designated as diets CM0, CM25, CM50, CM75 and CM100, respectively, based on percentage of protein replacement). The results showed that dietary CM significantly affected the coefficient of total apparent digestibility (CTTAD) of protein and phosphorus. However, in the long-term, it did not impact fish growth. Growth rates within male and female groups were not significantly affected by the dietary CM replacement after the 3 years of feeding. Gossypol-related mortality was not found in the fish during the 3 years of feeding. Muscle gossypol concentrations after 3 years were significantly different among groups and corresponded to the dietary concentration. Less than 1.0 μg gossypol/g wet basis of muscle was found in the highest dietary CM group. In males, the concentration of steroid hormones and the reproductive performances including sperm concentration, motility and eyed-stage embryo survival in the third year of this study were not significantly affected by the dietary CM inclusion. In contrast, female rainbow trout fertility and plasma testosterone levels were negatively affected by complete replacement of FM protein with CM protein. The findings based on this long-term study suggest that high dietary CM supplementation up to 58.8/100 g or complete substitution of FM protein does not impair growth of both sexes and reproductive performance of male rainbow trout, and that humans can safely consume the fillet produced by fish fed CM-containing diets.

Rainbow trout (Oncorhynchus mykiss) males were fed, during a 9-month period, five experimental diets where fishmeal proteins were gradually replaced with cottonseed meal (CS) proteins (0, 25, 50, 75 and 100%; diets 1–5, respectively). This study was carried out to evaluate the action and tissue concentrations of gossypol. Growth performance of fish was not affected with the increasing levels of CS in the diets. Haemoglobin concentration and haematocrit significantly decreased in fish fed 100% CS compared with other dietary treatments. Gonadosomatic index, plasma sex steroids (testosterone, 11-ketotestosterone and 17,20β-dihydroxy-4-pregnen-3-one) and sperm characteristics (concentration, motility, protein concentration and lactate dehydrogenase activity) were not negatively affected by increasing levels of CS. For the first time, a comprehensive study of gossypol concentrations, total and (+) and (−)-enantiomers, in several tissues (liver, blood plasma, spermatozoa and seminal plasma) was performed. The concentrations of both (+) and (−)-enantiomers significantly increased with increasing levels of CS in the diet. The highest concentrations were found in the liver of fish fed diet 5 (185 ± 18 μg g−1). In blood plasma, the concentrations of total gossypol were 10 times lower than the one found in the liver, but 10 and 100 times higher than the concentrations in the spermatozoa and seminal plasma, respectively. In all tissues studied, the concentration of (+)-enantiomer was higher than the (−)-enantiomer. The ratio (−)-enantiomer/total gossypol did not change significantly with the increasing levels of CS in the diet. The results of the present study indicate that CS can be used over a period of 9 months to replace fishmeal proteins completely without compromising growth and reproduction of rainbow trout males.

Muskellunge Esox masquinongy eggs activated with UV-irradiated yellow perch Perca flavescens sperm were subjected to heat shock at 31 ± 1° C for 6 min, 20 min after fertilization in three experiments. Survival at eyed stage was 1·7 ±1·6, 6·8 ± 4·8 and 2·3 ±0·5% in experiments I, II and III, respectively. After rearing the gynogenetic muskellunge in troughs and then in ponds, the sex ratio of gynogens in experiment I did not significantly (P>0·05) differ from the expected ratio 0:1 (male: female), however, one male and one intersex were observed. In experiments II and III, the sex ratio of gynogens differed significantly from expected (0 : 1). Three months after hatching, the growth of females did not significantly (P>0·05) differ among control and gynogenetic groups, whereas male growth was significantly (P

This study examined the changes in plasma steroids during natural (Experiment 1) and induced (Experiment 2) final maturation in yellow perch Perca flavescens. In experiment 1, ovulating yellow perch were stripped of eggs and blood samples collected to determine the concentrations of testosterone (T), estradiol-17β (E2), and 17,20β-dihydroxy-4-pregnen-3-one (17,20βP). Eggs from individual females were weighed and fertilized. Fertilization rate was determined at the embryo eyed stage. In experiment 2, females were randomly assigned to one of the following treatment groups: (1) saline (0.7% NaCl), (2) des-Gly10[D-Ala6] LHRH-ethylamide (100 μg LHRHa/kg), and (3) LHRHa plus 17,20βP (100 μg LHRHa/kg + 2 mg 17,20βP/kg). Fish were injected intraperitoneally with two doses at a two-day interval. Blood was collected prior to injections and at the time of ovulation/spawning and concentrations of T, E2, and 17,20βP (free and conjugated) were determined. In experiment 1, low concentrations of 17,20βP were recorded at spawning. In experiment 2, all surviving fish injected with LHRHa (5 of 5) released their eggs spontaneously during the week following injections. None of the surviving control fish (0 of 5) ovulated during this period, whereas only 1 of 3 surviving fish injected with LHRHa + 17,20βP released eggs. In the control group, concentrations of E2 and 17,20βP did not show significant differences over the experimental period, whereas plasma T concentrations increased significantly. In fish injected with LHRHa, the concentrations of T and 17,20βP increased significantly after the first injection but then declined at ovulation/spawning. It also appears that 17,20βP was conjugated to its sulfated form. Mortality reached 62.5% in the group injected with LHRHa + 17,20βP indicating that this treatment was severe. Thus, LHRHa alone appears highly effective in inducing ovulation in yellow perch.

Five experimental diets containing increasing proportions of cottonseedmeal (CSM) protein (0, 25, 50, 75 and 100%; diets 1 to 5, respectively) toreplace fish meal (FM) protein were formulated for intensive culture of tilapiaOreochromis sp. Each diet was fed to three replicategroupsof fish (mean weight ± SE = 11.3 ± 3.9 g) in30L aquaria connected as a closed recirculating-water system andmaintained at 27 ± 1 °C. Fish were fed three times adayby hand at a rate of 3% of body weight during four weeks, after which thefeeding rate was gradually decreased to reach 1.5% at 16 weeks. Thesubstitutionof 75 and 100% of FM proteins by CSM proteins resulted in significantly lowerbody weights in both sexes. In both sexes, gonadosomatic indexes and plasmaconcentrations of sex steroids (testosterone, 11-ketotestoterone,estradiol-17β and 17,20β-dihydroxy-4-pregnen-3-one) were notsignificantly different among dietary treatments. The concentration ofgossypol,an antifertility agent contained in CSM, was measured in reproductive tissues.The total gossypol concentration in the testis was consistently lower than thatmeasured in the ovaries of the same group. Moreover, in both sexes, theconcentration of the (+)isomer of gossypol was always higher than that of(−)isomer. The total gossypol concentrations in testes increasedsignificantly with the increase of CSM in the diet. The highest levels of the(+)isomer (7.64 ± 0.62 μg g−1)were found in the testes of fish fed diet 4, whereas the (−)isomerreached its highest values in the testes of fish fed diet 5. The highest levelsof both enantiomers of gossypol were found in the ovaries of fish fed diet 4(14.2 ± 2.7 and 5.6 ± 1.5 μg g−1for (+) and (−)isomers, respectively). In both sexes, thehistological analysis of the gonads did not reveal differences among the fishfed different levels of CSM. Although CSM at any levels did not affect thereproductive parameters examined in this study, it cannot be used to substitutemore than 50% of FM since at higher levels growth of tilapia was compromised.

Acrosin activity and semen quality (sperm concentration, ejaculate volume and number of spermatozoa) were assessed from March 1997 to March 1998 in semen of Large White, Pietrain and Duroc Pietrain boars. Semen quality varied with season, including high production of spermatozoa in autumn and winter and low production in summer. Semen quality also differed across breeds. Acrosin activity of boar spermatozoa was not affected by breed (range 3.16‐3.32 mU/10 6 spermatozoa), but exhibited distinct seasonal changes. Monthly changes in acrosin activity were parallel to changes in number of sperm in the ejaculate from November to March. On the other hand, dramatic changes in acrosin activity between July and October (range 1.85‐4.59 mU/10 6 spermatozoa) were not paralleled by similar changes in number of ejaculated sperm. These fluctuations in acrosin activity may reflect either changes in sperm acrosin production or disturbances to sperm membranes, probably related to effects of high summer temperatures during spermatogenesis. Results confirmed seasonal and breed-related differences in boar semen quality characteristics. © 2000 Elsevier Science B.V. All rights reserved.

We evaluated five practical diets in which 0%, 25%, 50%, 75%, and 100% (dietary treatments 1‐5) of fish meal protein was replaced by solvent-extracted cottonseed meal protein. Adult rainbow trout (initial average weight 247 6 8 g) were fed the diets over a period of 131 days during which a general 2fold body weight increase occurred. The total diet gossypol concentration (free and protein-bound) showed a gradual increase with increased cottonseed meal substitution. Blood samples were collected on Days 0, 64, 112, and 131 for hematological and steroid hormone determination in plasma of males and females. Hemoglobin content was significantly reduced in fish from treatment 5 (7.9 6 0.3 g/dl) in comparison to treatments 1‐3 (10.3‐10.9 g/dl). After 112 and 131 days of feeding, testis weights, concentrations of testosterone, and 11-ketotestosterone were elevated in fish from dietary treatments 2 and 3 in comparison to control and diets 4 and 5. On Day 71, sperm were collected from 6 fish per dietary treatment to assess sperm quality. No significant differences in sperm concentrations (7.2‐9.8 3 10 9 /ml), motility (78‐89%), and standardized (300 3 10 5 sperm/egg) fertilizing ability (18.9‐22.6% hatched embryos) were found. Total gossypol concentrations in blood plasma differed significantly among treatments, and the levels were among the highest ever recorded in animals fed cottonseed-supplemented diets (2.9 6 0.2, 11.7 6 4.1, 21.7 6 1.4, and 29.9 6 3.9 mg/ml, for treatments 2‐5, respectively). The major portion of gossypol in blood plasma was protein-bound (81‐93%). This was in contrast to minute amounts of gossypol present in seminal plasma, mostly in free form (0.02‐0.18 mg/ml), which indicates the presence of a barrier between general circulation and the testis with respect to gossypol distribution in lower vertebrates. Thus, the reproductive parameters of male rainbow trout examined in this study were not significantly affected by feeding cottonseed meal for 131 days.

Oral administration of 17α-methyltestosterone (MT) was used to induce masculinization of sexually undifferentiated muskellunge, Esox masquinongy. Three groups of muskellunge (mean weight, 2.5 ± 0.6 g) were submitted to MT treatment (15 mg of MT/kg) for 60 days. An additional one group was used as a control (hormone-free diet). Food was distributed over a 10-h period by using automatic belt feeders. Blood was sampled in both control and treated fish at different intervals during and after feeding: before (0 h), at 3 h, 6 h, and cessation of feeding (10 h), and after a fast of 22 h (32 h). MT had no significant effect on growth and survival in muskellunge 6 months after the treatment. Concentrations of plasma MT increased during the feeding period and reached their maximum levels 6 or 10 h after starting feeding. This rapid increase of MT indicated a rapid absorption of this steroid. Plasma MT levels then declined and reached a radir by 22 h after cessation of feeding, suggesting that MT is rapidly metabolized and excreted. The profiles of plasma testosterone during the MT treatment did not differ significantly between control and MT-treated groups. During and after the MT treatment, the concentration of plasma testosterone did not differ significantly between control and MT-treated groups. Moreover, no sexual dimorphism of testosterone levels was observed. Six months after treatment, the sex ratio in MT-treated groups (33% males, 62% females, and 5% intersex) was opposite to control (70% and 30%, respectively) and differed significantly. This suggests that at 15 mg of MT/kg over 60 days, a paradoxical feminization took place.

Previously, we reported that administration of prolactin (PRL) during the early luteal phase in sows increases plasma progesterone concentrations. In the current study, we searched for the mechanisms by which PRL exerts this luteotrophic effect. The objectives of the study were (1) to examine the effect of PRL and/or low-density lipoproteins (LDL) on progesterone production by porcine luteal cells derived from early corpora lutea, and (2) to assess the ability of PRL to activate phosphoinositide-specific phospholipase C (PI-PLC) and protein kinase C (PKC) in these luteal cells. Ovaries with early corpora lutea (day 1-2 of the oestrous cycle) were obtained from the slaughterhouse. Progesterone production by dispersed luteal cells was measured after treatment with PRL, phorbol 12-myristate 13-acetate or inhibitors of PKC in the presence or absence of LDL. LDL increased progesterone concentration in the incubation medium (304.5 vs 178.6 ng/ml in control, P0.05). Staurosporine, a PKC inhibitor, inhibited progesterone secretion stimulated by the combined action of LDL and PRL; however, such inhibition was not demonstrated when cells were treated with the PKC inhibitor, H-7. PKC activation was assessed by measuring the specific association of [H]phorbol dibutyrate (H-PDBu) with luteal cells after treatment with PRL or ionomycin (a positive control). PRL and ionomycin increased H-PDBu-specific binding in early luteal cells by 28+/-5.5% (within 5 min) and 70.2+/-19.3% (within 2 min) over control binding respectively (P

The luteolytic effects of prostaglandin F2 alpha (PGF2 alpha) are thought to be mediated in part by the promotion of an increasingly oxidative cellular environment. Loss of antioxidants is one mechanism by which PGF2 alpha might induce or exacerbate oxidative damage within the corpus luteum. This study was performed to establish whether depletion of vitamin C is an acute effect of PGF2 alpha on the pig corpus luteum and to gain insight into the mechanism of luteal vitamin C loss at luteolysis. Gilts (n = 4) were anaesthetized and both utero-ovarian veins and an ear vein were catheterized. Each corpus luteum on the treated ovary received an intraluteal injection of PGF2 alpha (1 microgram) followed by a sustained release implant containing 100 micrograms of the prostaglandin. The other ovary served as the control and each corpus luteum received corresponding volumes of injection vehicle and blank implant. Blood was collected from the ear vein and both utero-ovarian veins every 15 min beginning 15 min before the onset of treatment. Collection of blood stopped when animals were ovariectomized and corpora lutea were collected at 2 h after treatment. Progesterone and vitamin C (ascorbate) concentrations were measured in tissue and plasma samples. PGF2 alpha-treated luteal tissue had similar progesterone, but significantly lower ascorbate, concentrations when compared with control corpora lutea. PGF2 alpha treatment resulted in a rapid and sustained increase in plasma ascorbate within the treatment-side utero-ovarian vein, while the control utero-ovarian vein and ear vein showed little change in plasma ascorbate during the experimental period. No effect of PGF2 alpha on plasma progesterone was evident. This finding suggests that PGF2 alpha depletes the pig corpus luteum of vitamin C by inducing secretion of the vitamin into the bloodstream. Further studies are necessary to determine whether the depletion of vitamin C that is induced by PGF2 alpha contributes to the demise of the pig corpus luteum.

Plasma steroid concentrations in two and three year-old male yellow perch maintained under two different photothermal regimes were investigated. Initially, all fish kept indoors were exposed to the same water temperature (22 °C) and photoperiod (15L:9D). By the end of August, following the first sampling, fish were exposed to different photothermal regimes. Groups A2 (2 year old) and A3 (3 year old) were maintained under photothermal conditions similar to those of southern Ohio. Groups B2 (2 year old) and B3 (3 year old) were exposed to a condensed light/temperature regime designed to accelerate maturation. Testosterone (T) was the major circulating androgen in all groups. In regime A fish, plasma concentrations of 11-ketotestosterone (11KT) and T were very low in August, increased in October and remained elevated until March. In regime B plasma androgens were high until February and then dropped abruptly in March. The elevated circulating levels of 11KT and T were associated with production of sperm. The highest sperm concentration in the groups A3 and B3 was observed in February and December, respectively. There were no major differences in profiles and levels of plasma steroids between two age categories within each photothermal regime. These data indicate that the compression of the photothermal cycle accelerated both the occurrence of the low postspawning levels of circulating steroids and the completion of milt production. Higher sperm concentration observed in B3 group earlier in the season compared to A3 group also support the notion that the condensed photothermal cycle accelerated gonadal maturation. It appears that modification of the environmental cues may be a useful tool for manipulation of reproductive processes in male yellow perch.

Two synchronization protocols were tested for lactating dairy cows and heifers. Nulliparous dairy heifers (13 to 23 mo; n = 155) and primiparous and multiparous dairy cows (60 to 289 d postpartum; n = 310) were assigned randomly to two treatments. Controls received 25 mg of PGF2 alpha and were artificially inseminated according to the a.m.-p.m. rule following detected estrus. All controls that were not detected in estrus were injected with 25 mg of PGF2 alpha at 14-d intervals until artificial insemination (AI) at a detected estrus or until timed AI at 72 to 80 h after a third sequential injection of PGF2 alpha. Treated cows and heifers received a protocol that used GnRH and PGF2 alpha to synchronize ovulation (Ovsynch). Cows and heifers that were treated with Ovsynch were injected i.m. with 100 micrograms of GnRH at a random stage of the estrous cycle. Seven days later, cows and heifers in this group received 25 mg of PGF2 alpha followed by a second injection of 100 micrograms of GnRH 30 to 36 h later. Subsequently, the treated cows and heifers received AI 16 to 20 h after the second injection of GnRH. Pregnancy rates per AI were similar (38.9% vs. 37.8%) for control cows and cows treated with the Ovsynch protocol, respectively. However, pregnancy rate per AI was greater for control heifers (74.4%) than for heifers treated with Ovsynch (35.1%). Evaluation of serum progesterone concentrations at each hormonal injection indicated that the first injection of GnRH synchronized luteal function of lactating dairy cows but not of heifers. In summary, one fixed-time AI at a synchronized ovulation provided similar pregnancy rates per AI as did AI following the a.m-p.m. rule after estrus had been induced by PGF2 alpha in lactating cows, but the fixed-time AI was not effective for heifers because of the lack of synchronization.

An understanding of the mechanisms involved in the regulation of reproductive processes in yellow perch is fundamental for intensive culture of this commercially important, freshwater, perciform fish. This paper describes the annual reproductive cycle of female and male perch. It presents the current state of knowledge on the regulation of ovarian steroidogenesis, final maturation of oocytes and ovulation in this species. Moreover, data concerning biochemistry of spermatozoa and seminal plasma as well as testicular steroidogenesis are summarized. In addition to endocrinological factors, the involvement of environmental (photoperiod and temperature) cues in the regulation of yellow perch reproduction is reviewed. Finally, practical aspects of controlling perch reproduction are outlined.

The current study was designed to examine the effects of prostaglandin (PG) E2 on progesterone production by primate luteal cells collected during the late luteal phase. PGE2 inhibited basal and human chorionic gonadotrophin (HCG)-stimulated progesterone production (P < 0.01) in late luteal phase corpora lutea. The ability of PGE2 to activate a second messenger system (phosphatidylinositol pathway) in corpora lutea of rhesus monkeys was also assessed. PGE2 significantly increased the accumulation of inositol phosphates (P < 0.05). This stimulation was not apparent in the early luteal phase but was manifested in the mid-late luteal phase. PGE2 also caused a rapid, yet transient, increase (P < 0.01) in intracellular free calcium ion concentrations ([Ca2+]i) in a large proportion of primate luteal cells. The proportion of luteal cells that responded to PGE2 with an increase in [Ca2+]i was smaller (P < 0.05) in corpora lutea collected during the early luteal phase (12%) in comparison with those collected during the latter half of the luteal phase (63-66%). Changes in [Ca2+]i in response to PGE2 were similar in small and large luteal cells. This study demonstrates that PGE2 activates elements of the phosphatidylinositol pathway in primate corpora lutea. This activation is augmented as the luteal phase progresses. Thus, the inhibitory effects of PGE2 on luteal progesterone production observed in the late luteal phase are associated with activation of elements of the phosphatidylinositol pathway.

Diabetic heart disease is a distinct clinical entity that can progress to heart failure and sudden death. However, the mechanisms responsible for the alterations in excitation-contraction coupling leading to cardiac dysfunction during diabetes are not well known. Hyperglycemia, the landmark of diabetes, leads to the formation of advanced glycation end products (AGEs) on long-lived proteins, including sarcoplasmic reticulum (SR) Ca(2+) regulatory proteins. However, their pathogenic role on SR Ca(2+) handling in cardiac myocytes is unknown. Therefore, we investigated whether an AGE cross-link breaker could prevent the alterations in SR Ca(2+) cycling that lead to in vivo cardiac dysfunction during diabetes. Streptozotocin-induced diabetic rats were treated with alagebrium chloride (ALT-711) for 8 weeks and compared to age-matched placebo-treated diabetic rats and healthy rats. Cardiac function was assessed by echocardiographic examination. Ventricular myocytes were isolated to assess SR Ca(2+) cycling by confocal imaging and quantitative Western blots. Diabetes resulted in in vivo cardiac dysfunction and ALT-711 therapy partially alleviated diastolic dysfunction by decreasing isovolumetric relaxation time and myocardial performance index (MPI) (by 27 and 41% vs. untreated diabetic rats, respectively, P0.05). In cardiac myocytes, diabetes-induced prolongation of cytosolic Ca(2+) transient clearance by 43% and decreased SR Ca(2+) load by 25% (P0.05); these parameters were partially improved after ALT-711 therapy. SERCA2a and RyR2 protein expression was significantly decreased in the myocardium of untreated diabetic rats (by 64 and 36% vs. controls, respectively, P0.05), but preserved in the treated diabetic group compared to controls. Collectively, our results suggest that, in a model of type 1 diabetes, AGE accumulation primarily impairs SR Ca(2+) reuptake in cardiac myocytes and that long-term treatment with an AGE cross-link breaker partially normalized SR Ca(2+) handling and improved diabetic cardiomyopathy.

Blood samples were taken at 0800 and 2000 hours each day from 6 horse mares for the duration of one estrous cycle, and from 6 mares for the first 35 days of pregnancy. Progesterone concentration was determined by radioimmunoassay. Data were subjected to repeated measures analyses of variance to compare pregnant vs nonpregnant animals and day and time of day. In addition, within group analyses were performed on data from Days 1 to 18 post ovulation in nonpregnant mares and from Day 1 to 35 post ovulation in pregnant mares. There was no significant effect of time (P=0.1383), and no interaction of time by group (P=0.3225), time by day (P=0.2524), or time by day by group (P=0.5354). Since morning and evening progesterone concentrations did not differ on any day within either group, an average daily progesterone concentration was calculated for each horse on each day. Comparison of daily mean progesterone concentrations (pregnant vs nonpregnant) revealed that pregnant mares did not have higher progesterone concentrations than nonpregnant mares until Days 16, 17 and 18 post ovulation (P There do not appear to be regular diurnal variations in serum progesterone concentrations in the horse mare, although substantial random variation in circulating progesterone can occur throughout the day.

The current study was designed to investigate the ability of prostaglandin F2 alpha (PGF2 alpha) to activate a second messenger system (phosphatidylinositol pathway) in corpora lutea (CL) of rhesus monkeys. Activation of this pathway was assessed by monitoring the hydrolysis of phosphatidylinositol to inositol phosphates. Since inositol triphosphate mobilizes intracellular Ca2+, intracellular free calcium concentrations ([Ca2+]i) were also assessed in individual cells by fura-2 fluorescence photometry. These responses to PGF2 alpha were measured in luteal cells collected from nonpregnant rhesus monkeys. CL were collected during the early (days 4-5 after estimated LH surge; n = 4), mid (days 8-9; n = 4), and late (days 13-14; n = 5) luteal phase and 1 day after in vivo hCG treatment (15 IU/dose, morning and evening), which began during the midluteal phase (n = 5). PGF2 alpha significantly increased the accumulation of inositol phosphates in all groups (P less than 0.05), except the midluteal phase (P = 0.07). The luteal sensitivity to PGF2 alpha, judged by phosphatidylinositol hydrolysis, was low in the early to midluteal phase compared to that in the late luteal phase and after in vivo hCG treatment. PGF2 alpha also caused a rapid, yet transient, increase in [Ca2+]i in a large proportion of primate luteal cells. The proportion of luteal cells that responded to PGF2 alpha with an increase in [Ca2+]i was smaller (P less than 0.05) in CL collected during the early luteal phase than in the other groups. Luteal progesterone production was inhibited by PGF2 alpha in CL collected after in vivo hCG. CL treated in vivo with hCG also displayed in vitro the largest increases in phosphatidylinositol hydrolysis and [Ca2+]i in response to PGF2 alpha. Therefore, this study demonstrates that PGF2 alpha is a potent activator of the phosphatidylinositol pathway in the primate CL. This activation is augmented as the luteal phase progresses and is influenced by in vivo hCG treatment. This study also provides evidence that the inhibitory effects of PGF2 alpha on progesterone production are associated with the activation of the phosphatidylinositol pathway.

The role of the phosphatidylinositol second messenger system in luteal regulation has not been extensively studied, particularly in the primate. The objectivesof thisstudy were (1) to further characterize the response of the primate CL to the calcium ionophore A23187, in terms of intracellularfreecalcium concentrations (lCa2l1) and progesterone (P) production; and (2) to assess the effectsof depleting,aswell as elevating,availablecalcium on lutealP and prostaglandin (PG) production. The response to A23187, in terms of [Ca’l, was measured by fura-2 fluorescence microscopy of single small and large lutcal cells. A23187 significantly increased lCa’4l in both cell types (p < 0.01). P production (basaland hCG-stimulated) by dispersed primate luteal cells incubated for various tunes (1-8 h) with and without A23187 was measured. Treatment with A23187 rapidly (within 12 h) attenuated (� < 0.05) the time-dependent increase in basal and hCG-stimulated P production. Luteal P and PG production following treatment with the calcium ionophore, lonomycin, alone or in combination with additional CaCl,,was alsomonitored. Treatment with ionomycin (p < 0.01) and CaCI2 (� < 0.01) inhibitedlutealP production. Incontrast,treatmentwith ionomycin stimulated (p < 0.01) lutealPG production. To determine the effects of Ca’� depletion on luteal function, P and PG production by cellsincubated for 2 and 8 h in the absence and presence of the Ca’�-chelatorEGTA was measured. Luteal production of both P and PG was inhibitedby 8-h treatment with EGTA. In conclusion, elevations in lCa’�l differentially regulate PG and P production in the primate CL, leading to elevated PG and suppressed P production in CL collected in the mid-luteal phase. Since thispattern of luteal PG and P production is also evident latein the lutealphase, the phosphatidylinositolpathway may be involved in regressionof the primate CL In contrast to the selectivealterationsin lutealfunctionfollowing increases in lCa21i, depletion of available calcium leads to suppression of both PG and P production. Thus, an optimal level of tCa2�] may be necessary for maximal luteal function.

The objective of this study was to characterize endometrial secretion (in vitro) of prostaglandin F (PGF), 15-keto-13,14-dihydro-prostaglandin F2 alpha (PGFM), prostaglandin E2 (PGE2), and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) on Day 5 following the first postpartum estrus of cows anticipated to have a short compared to a normal estrous cycle. Twenty-seven beef cows were randomly assigned into four groups. The Short Cycle (n = 6; control) and Short Cycle/Explant (n = 8; endometrial explants) groups had their calves weaned at 30-32 days postpartum. The Normal Cycle (n = 5, control) and Normal Cycle/Explant (n = 8; endometrial explants) groups received norgestomet (progestin) implants for 9 days beginning 21-23 days postpartum, and calves were weaned at implant insertion. Estrous cycle length (mean +/- SE; p less than 0.01) for the Short Cycle group was 11.5 +/- 1.9 days compared to 18.8 +/- 0.6 days for the Normal Cycle group. On Day 5 following the first postpartum estrus, cows in the Short Cycle/Explant and Normal Cycle/Explant groups were hysterectomized, and endometrial explants were incubated in Earle's Balanced Salt solution/Medium 199 for 90 min with or without arachidonic acid (AA) in the presence of three levels of oxytocin. Mean concentrations of PGF and PGFM were combined to obtain a value for total PGF. Concentrations of total PGF, PGE2 (from explants without AA treatment), and 6-keto-PGF1 alpha in medium of the Short Cycle/Explant group were higher (p less than 0.01) than in medium of the Normal Cycle/Explant group.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and characterization of biological changes which occur near the time of ovulation may eventually provide improved means for determining the timing of this event. A rise in internal body temperature is associated with estrus in dairy cows, but temporal relationships between this temperature rise and other periovulatory events require further investigation. The objectives of this study were: (1) to determine the temporal relationships among changes in serum concentrations of luteinizing hormone (LH), estradiol, progesterone, and changes in body temperature that occur during the periovulatory period, and (2) to assess the consistency among individuals of these temporal relationships by comparing variabilities among cows of intervals between periovulatory events. Vaginal temperature was monitored in eight, nonpregnant, lactating dairy cows using temperature-sensing radiotransmitters. Serum concentrations of progesterone, LH, and estradiol were determined by radioimmunoassay (RIA). Temperature spikes were defined as sustained increases in temperature of ≥ 0.3°C. Intervals between various periovulatory events were calculated (e.g., LH peak to onset of temperature spike, estradiol peak to LH peak, etc.). Variances of these intervals among cows were then compared using an F-test and were considered significantly different if P was

Increases in internal body temperature have been observed near estrus in cattle. In our companion paper, an estrual rise in vaginal temperature occurred within a consistent interval from the LH surge. However, the relationship of this rise in temperature to ovulation was not determined. The objectives of this study were to: (1) characterize the temporal relationship between the estrual increase in body temperature and ovulation in the heifer, and (2) assess the consistency of this temporal relationship among individuals by comparing it to the consistency of intervals between other periovulatory events. Body temperature was monitored automatically and recorded every 15 min with intravaginal radiotransmitters in post-puberal heifers. Temperatures were averaged hourly. To monitor the demise of the corpus luteum and anticipate estrus, progesterone (P) concentrations were monitored. Blood samples were collected daily, beginning on day 16, until serum P fell to ≤ 0.5 ng/ml. Samples were then collected every 4 h until ovulation. Serum concentrations of estradiol-17β (E) and luteinizing hormone (LH) were measured in these samples. Serum concentrations of P, E and LH were determined by radioimmunoassay. Hourly temperature averages were compared to the previous 3 days' hourly temperature averages for each hour of the day. A significant ( P P > 0.10, F -test): E peak to onset of a TS (1.00 ± 5.90 h, mean ± s.d., n = 6), LH peak to onset of a TS (0.29 ± 5.22 h, n = 7), E peak to TO (22.30 ± 3.92 h, n = 6), LH peak to TO (21.43 ± 4.31 h, n = 7), and onset of a TS to TO (21.14 ± 6.07 h, n = 7). In conclusion, intervals between periovulatory events had similar variances among heifers. Since the interval between the LH peak to ovulation is considered a repeatable periovulatory relationship, all periovulatory intervals in the current study can be considered repeatable. As such, relative to other periovulatory intervals, the interval between the onset of a TS to TO is consistent. Therefore, the onset of a TS, as monitored by radiotelemetry, may be as reliable a predictor of ovulation as the LH surge or other periovulatory events.

Basal and calcium ionophore (CaI)-influenced production of prostaglandins (PGs) by corpora lutea (CL) destined to be normal or short-lived were compared. Ovulation was induced in 24 lactating beef cows with human chorionic gonadotropin (hCG, 1000 IU) administered between 35 and 40 days postpartum. Ten cows received norgestomet implants for 9 days prior to induced ovulation (Normal CL) and 14 served as untreated controls (Subnormal CL). Five cows in each treatment were unilaterally ovariectomized on Day 6 (Day 0 = day of hCG administration) and CL were collected. Blood samples were collected daily through-out the experimental period from cows not ovariectomized. Plasma progesterone (P4) in ovary-intact animals indicated that short-lived CL were induced in 8/8 cows not pretreated with norgestomet, and normal luteal lifespan was observed in 4/5 implanted cows. Dispersed luteal cells were incubated for 8 h with 0, 0.05, 0.5, or 5 microM CaI (A23187). Incubation media were analyzed for P4, PGF2 alpha, 6-keto-PGF1 alpha (PGI), and PGE2. The weight, cell number, and basal or CaI-influenced production of P4 did not differ between Normal CL and Subnormal CL. Basal production of PGF2 alpha, PGI, and PGE2 was higher in Subnormal CL than in Normal CL (p less than 0.05). In response to 0.05 microM CaI, PGF2 alpha was stimulated in Subnormal CL (p less than 0.01), while PGI (p less than 0.05) and PGE2 (p less than 0.1) were increased in Normal CL. Production of PGs was reduced by 5 microM CaI in Subnormal CL (p less than 0.01), but not in Normal CL.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell cultures were established from the skeletal muscle tissue of 6-13 months old rainbow trout and 12-14 months old yellow perch. Approximately 27,000 +/- 5,000 cells/g (trout; N = 5) and 5,000 +/- 1,200 cells/g of tissue (perch; N = 4) were obtained. Isolation and propagation were qualitatively greater for both species when the cells (younger fish producer more cells than older fish) were exposed to DMEM + 15% FBS, rather than L-15 + 15% FBS, at 20 degrees C (trout) and at 24 degrees C (yellow perch). Two morphologically distinct cell types were observed in cultures of both species, some of which eventually formed very small myotubes, which displayed immunocytological reactivity for myogenin, myosin heavy chain, and alpha-actinin; the second population of cells remained unstained. Successful cryopreservation was achieved using a 5% DMSO and 95% serum mixture, but post-thawing viabilities were low 5-27% (trout) and 14-30% (perch). Further research is needed in order to determine cell type specificity of isolated cells.

Our objective was to determine whether progesterone (P4) supplementation during an Ovsynch protocol would enhance fertility in lactating dairy cows. Lactating dairy cows (n = 634) at 6 locations were assigned randomly within lactation number and stage of lactation to receive the Ovsynch protocol [OVS; synchronization of ovulation by injecting GnRH 7 d before and 48 h after PGF(2alpha), followed by one fixed-time AI (TAI) 16 to 20 h after the second GnRH injection] or Ovsynch plus a controlled internal drug release (CIDR) P4-releasing insert for 7 d, beginning at the first GnRH injection (OVS + CIDR). Blood was sampled to quantify P4 10 d before the first GnRH injection, immediately before the first GnRH injection, at the time of CIDR removal, before the PGF(2alpha) injection (1 to 2 h after CIDR insert removal), and 48 h after the PGF(2alpha) injection to determine cyclicity status before initiation of treatment, luteal status at the PGF(2alpha) injection, and incidence of luteal regression. Overall, conception rates at 28 (40 vs. 50%) and 56 d (33 vs. 38%) after TAI differed between OVS and OVS + CIDR, respectively; but a treatment x location interaction was detected. Compared with OVS, pregnancy outcomes were more positive for OVS + CIDR cows at 4 of 6 locations 28 d after TAI and at 3 of 6 locations 56 d after TAI. An interaction of luteal status (high vs. low) before CIDR insert removal and PGF(2alpha) injection with pretreatment cycling status indicated that cows having low P4 at PGF(2alpha) injection benefited most from P4 supplementation (OVS + CIDR = 36% vs. OVS = 18%), regardless of pretreatment cycling status. Pregnancy loss between 28 and 56 d after TAI was greater for noncycling cows (31%) compared with cycling cows (16%). Pregnancy loss for cows receiving P4 (21%) did not differ from that for cows not receiving P4 (21%). Supplementation of P4, pretreatment cycling status, and luteal status before PGF(2alpha) injection altered follicular diameters at the time of the second GnRH injection, but were unrelated to pregnancy outcomes. Incidence of multiple ovulation was greater in noncycling than in cycling cows. Further, cows having multiple ovulations had improved pregnancy outcomes at 28 and 56 d after TAI. In summary, a CIDR insert during the Ovsynch protocol increased fertility in lactating cows having low serum P4 before PGF(2alpha) injection. Improved pregnancy outcomes were observed at some, but not all locations.

We investigated the capacity of ovaries of yellow perch to produce steroid hormones in vitro and the ovarian response to gonadotropin and phorbol ester during the annual reproductive cycle. The effects of photothermal manipulation on perch gonadal steroidogenesis and its regulation have also been examined. Initially, all females kept indoors were exposed to the same water temperature and photoperiod. By the end of August, following the first sampling, fish were submitted to different photothermal regimes. Group A was maintained under photothermal conditions characteristic for southern Ohio. Group B was submitted to a condensed light/temperature regime designed to accelerate physiological changes that depend on photothermal stimuli. In group A, basal in vitro production of ovarian estradiol (E2) was the highest in October and November, and hCG significantly stimulated E2 secretion during the entire period of vitellogenesis (October-January). In this group, the highest production of basal testosterone (T) was observed before spawning. hCG-stimulated production of T was highest at the beginning of vitellogenesis. Gonadotropin stimulated T production before spawning, a time when gonadotropin was unable to stimulate E2 production. Phorbol ester (PDBu) stimulated E2 and T production during vitellogenesis at the same time points as hCG did (E2: December, January; T: December). hCG-stimulated T production was not mimicked by PDBu in April. Condensing of the photothermal cycle resulted in diminished ovarian production of E2 during vitellogenesis. Moreover, the fish submitted to a condensed photothermal cycle demonstrated augmented T production during the postvitellogenic stage of ovarian development. Ovaries of group B did not respond to PDBu. Generally, the seasonal fluctuations in ovarian capacity to produce E2 and T as well as in gonadal responsiveness to gonadotropin observed in female yellow perch illustrate the dynamic nature of ovarian endocrine function. The lack of response to gonadotropin with regard to E2 production prior to spawning is not due to insensitivity to gonadotropin, but rather due to some deficiency in steroidogenesis (e.g. reduced aromatase activity). It appears also that ovarian steroidogenesis and its regulation are dependent on annual changes of photothermal conditions.

We optimized a clinical assay developed for measuring total acrosin activity for mammalian and fish semen for use in turkey spermatozoa. The main modifications included dilution of semen to a final concentration of 25 to 1000 × 103 spermatozoa, an increase of Triton X-100 concentration to 0.05% and 1 hr preincubation without substrate. Acrosin activity in turkey spermatozoa was much higher than in human spermatozoa (about 100-times) but similar to that of boar sperm. To optimize this assay for turkey spermatozoa, it was necessary to use higher Triton X-100 concentrations in the reaction mixture. There was a better catalytic efficiency at higher temperatures and a special requirement for a preincubation period for proacrosin activation. We observed high inhibition of acrosin activity by zinc added during preincubation (90% at 0.01 mM of zinc chloride). Benzamidine also inhibited turkey acrosin, and the extent of inhibition was similar for the incubation or preincubation period. When zinc ions were added during incubation, this inhibition was lower (24%). The results suggest that zinc influences proacrosin activation of turkey spermatozoa. This influence may be important for successful long-term storage of spermatozoa in the hen's oviduct.

The ontogeny of ascorbic acid synthesis and its concentration in fetal pigs from mid- to late gestation, and the effect of birth order and premature or normal delivery ages were evaluated. In Experiment 1, fetal pigs were collected from three sows at 60, 80, 100, 107 and 111 d of development. Liver L-gulono-gamma-lactone oxidase (GLO) activity and ascorbic acid concentration were measured. High liver GLO activity in fetal liver occurred at 60 d but declined as pregnancy advanced (P < 0.01), whereas ascorbic acid concentration increased (P < 0.01). Experiment 2 evaluated ascorbic acid synthesis and concentration in neonates born early (1st and 2nd) or late (7th and 8th) in the birthing sequence, or when born 2 d prematurely vs. the normal delivery age. Pigs born early in the birthing sequence (P < 0.01) and those born at the natural delivery age (P < 0.05) had higher liver ascorbic acid concentrations, but liver GLO activity did not differ among groups. Sows were killed at each period; liver GLO activity was constant during gestation but increased postpartum (P < 0.01). Liver ascorbic acid concentration was constant during gestation, except for a decline during late gestation, and increased postpartum (P < 0.05). These results suggest that more ascorbic acid was transferred from the dam to the fetuses as pregnancy advanced, possibly suppressing fetal GLO activity. Thus, fetal liver GLO activity was the primary source of ascorbic acid during early fetal development, but more fetal ascorbic acid was transferred from the dam during later pregnancy.

We investigated the role of gossypol isomers binding to blood plasma, seminal plasma and spermatozoa to elucidate gossypol anti-fertility action in the teleost fish, rainbow trout (Oncorhynchus mykiss). Growth and hematological indicators of males were depressed when fish meal protein in diets was completely replaced with cottonseed meal. The cottonseed meal contained equal proportions of (-) (47.8+/-1.6%) and (+) gossypol isomers. Concentrations of spermatozoa were decreased with increasing proportions of gossypol in diets (from 0.22% to 0.95%); however, sperm motility and fertilizing ability were not affected. In contrast to mammals, steroid hormone concentrations were not suppressed in fish given diets with gradual increase of gossypol level. Gossypol concentrations were 100-fold higher in blood plasma than in seminal plasma, confirming a barrier in gossypol transfer between the general circulation and the testis. Spermatozoa accumulated predominantly (+) enantiomer (65-75%) with decreasing proportions as dietary gossypol concentrations increased. Spermatozoa bound most of the gossypol contained in the semen; however, this did not result in impairment of the sperm motility apparatus. Teleost fish sperm rely on ATP stores that accumulate during maturation as a source of energy during activation. In addition, the duration of sperm movement is short in these fish. As such, we hypothesize that the major action of gossypol on mammalian sperm, which is uncoupling of oxidative phosphorylation, does not impair the energy supply required for flagellar beating in fish spermatozoa.

Male sea lampreys Petromyzon marinus were injected with different doses of gossypol acetic acid in an attempt to sterilize them for use in a program for controlling the sea lampreys through the release of sterile males. Two lots of sea lamprey were used in these experiments. The first lot was divided into three groups and fish were injected intraperitoneally (i.p.) with 0.2 ml 50% ethanol as a control group or with gossypol suspended in ethanol at 100 and 200 mg/kg. The second lot was also divided into three groups and fish were either injected i.p. with vehicle as controls or gossypol at 25 and 50 mg/kg. Sperm weight, concentrations and motility were recorded after 31, 36 and 40 days or 24, 28 and 33 days in lots 1 and 2, respectively. Blood was collected from the caudal vessel prior to injections with gossypol and after 40 or 33 days in lots 1 and 2, respectively. Plasma levels of estradiol-17beta (E2), testosterone (T), progesterone (P) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20betaP) were measured by radioimmunoassay. At the end of the experiment, the testis were removed and fixed in Bouin's solution for histological examination. High mortality was observed at the day of injection in the group treated with 200 mg/kg (84.6%), 100 mg/kg (41.7%), and 50 mg/kg (25%). Sperm concentrations were higher in control fish in comparison to most of the treated groups during the first sperm sampling (day 31 or 24), but then differences disappeared. At each sampling, sperm motility was higher in control groups than in treated groups and significant differences were observed (e.g. between control and 50 mg gossypol/kg). Fertility, evaluated at optimized sperm/egg ratio (5 x 10(4) sperm/egg) did not differ among treatments and controls. Changes in mean plasma sex steroid levels in the various treated groups were not significant, but a trend of decreasing plasma E2 was observed with increasing dose of gossypol. The structure of the testis was examined at the end of the experiment using light microscopy and appeared to be unchanged in gossypol-treated lampreys as compared to that of control animals. These findings suggest that gossypol injection into lamprey interferes with some of the reproductive parameters. Although high mortality rate precludes a single i.p. injection of gossypol as a favorable mode of chemosterilization in the lamprey, it would be useful to reassess the means of transfer of gossypol to reproductive tissues. This notion is further supported by the results of an in vitro sperm toxicity assay which revealed that gossypol, at a dose of 100 microM, has an antifertility action in this species.

Ovarian tissues are thought to require ascorbate as an antioxidant and enzymatic cofactor for the processes of steroid and collagen synthesis. We measured the concentrations of total ascorbate and oxidized ascorbate (dehydroascorbate, DHA) in ovarian stroma, follicles and corpora lutea (CL) throughout the estrous cycle and pregnancy of the sow. Both total ascorbate and DHA concentrations were greatest in luteal tissue and lowest in ovarian stroma across all stages examined. Within the CL, total ascorbate levels were lowest during the early, early-mid, and late luteal phase and were elevated during the mid-luteal phase. Luteal total ascorbate concentrations were further elevated during early pregnancy and were comparable to mid-luteal phase concentrations during the remainder of gestation. Luteal DHA concentrations decreased from mid to late luteal phase, and were elevated throughout pregnancy. As the CL aged during the cycle, the DHA/total ascorbate ratio decreased and remained low throughout pregnancy. Total ascorbate concentrations in follicular tissue increased during the follicular phase and were lowest during the early luteal phase. The DHA concentrations and DHA/total ascorbate ratios in follicular tissue did not differ with stage. Total ascorbate and DHA concentrations in ovarian stroma were low and did not vary with stage. We conclude that periods of maximal luteal and follicular function are associated with increased concentrations of total ascorbate within the tissue. Furthermore, luteolysis appears to be associated with depletion of luteal ascorbate species.

Arachidonic acid (AA) is a precursor of metabolites known to affect the corpus luteum (CL) in many species, including primates. We have shown that some of these products (prostaglandins F2 alpha and E2) inhibit pro-gesterone (P4) production and activate the phosphatidylinositol (PI) pathway in CL of rhesus monkeys. A direct role of AA in luteal function has also been suggested. The current experiments were designed to investigate the effect of AA on P4 synthesis and to examine the ability of AA to activate the PI pathway in CL of rhesus monkeys. Basal and hCG-stimulated P4 production by luteal cells collected during the midluteal phase was measured after treatment with AA (1, 5, and 10 microM) or linoleic acid (1, 5, and 10 microM). Dispersed cells (50,000/tube) were incubated at 37 degrees C for 2 h. AA elicited a dose-dependent decrease in hCG-stimulated, but not in basal, P4 production. hCG-stimulated P4 production was reduced (P < 0.01) at AA doses of 5 microM (12.1 +/- 1.5 ng/mL) and 10 microM (8.6 +/- 1.8 mg/mL) to hCG alone (18 +/- 1.6 ng/mL). There was no significant effect of 1 microM AA (15.2 +/- 1.6). Response to linoleic acid was dissimilar and was not dose-dependent. Viability of cells was not affected by any treatment. Indomethacin, a prostaglandin synthesis inhibitor, and nordihydroguaiaretic acid, an inhibitor of lipoxygenase, did not interfere with the inhibitory effect of AA. Activation of the PI pathway was assessed by monitoring the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) to inositol phosphates and by monitoring increases in intracellular free calcium concentrations ([Ca2+]i) in individual cells. Moreover, the ability of AA to activate protein kinase C (PKC) in luteal cells was measured using a [3H]phorbol dibutyrate (PDBu) binding assay. AA did not alter PIP2 hydrolysis or [Ca2+]i, however, AA (10 microM) increased specific binding of [3H]PDBu to luteal cells (P < 0.05). We conclude that AA inhibits hCG-stimulated P4 production by primate luteal cells. AA exerts this action without being converted to prostaglandins or leukotrienes. This inhibition may be mediated through the activation of PKC. These results suggest a possible role for AA in the regulation of luteal function in primates, and that PKC-activation by AA may promote its effects.

Two-year old rainbow trout females were fed diets containing 0, 30, 110, 220, 440 and 870 mg kg-1 ascorbyl-2-monophosphate Mg+ salt (groups 1, 2, 3, 4, 5, and 6, respectively) from August until March. At the time of spawning (February–March) blood was sampled and the ovulating females were hand stripped. Estradiol (E2) and testosterone (T) concentrations in plasma, and ascorbic acid (AA) concentrations in plasma and eggs were determined. The mean plasma concentrations of T were higher in group 4, 5, or 6 than in group 2 or 3 (p < 0.05). Moreover, the average plasma concentration of T in fish fed the diets with AA level below National Research Council (NRC) recommendations (groups 1, 2 and 3) was significantly lower (p

The current study was designed to investigate the effects of gonadotropin on basal and prostaglandin (PG) F2 alpha-induced activity of the phosphatidylinositol pathway in corpora lutea (CL) of rhesus monkeys. Luteal progesterone production in vitro was significantly stimulated (P0.05) by human chorionic gonadotropin (hCG). Neither basal nor PGF2 alpha-induced phosphatidylinositol 4,5-bisphosphate hydrolysis was significantly influenced by hCG in CL of various ages (P0.10). Gonadotropin did induce a slight, yet sustained, increase (P0.05) in [Ca2+]i in approximately 70% of luteal cells. The maximal increase in [Ca2+]i in response to hCG (approximately 100 nM) was about one-tenth that induced by PGF2 alpha (approximately 1000 nM). hCG treatment did not alter (P0.10) the increase in [Ca2+]i induced by PGF2 alpha Treatment-induced changes in [Ca2+]i did not differ between small (17-21 microns) and large (23-28 microns) luteal cells. Therefore, luteolytic agents are more potent activators of the phosphatidylinositol pathway than luteotropins. This is consistent with the hypothesis that the phosphatidylinositol pathway is involved in primate luteal regression. The inability of hCG to acutely alter the responsiveness of this pathway to PGF2 alpha suggests that CG may rescue the CL of early pregnancy via a mechanism other than direct inhibition of the luteolytic actions of PGF2 alpha.

During development of the corpus luteum (CL), the numbers of luteinizing hormone (LH) receptors increase. Cultured bovine luteal cells from developing and mature CL were used to examine the influence of progesterone (P4) on this receptor. CL were obtained from dairy cows during the early or middle phase of the estrous cycle. In early CL, the number of receptors per cell was increased by exogenous progesterone treatment but there was no effect on receptor numbers in cells from midcycle CL. Binding affinities did not change with respect to age or treatment. Forskolin elevated endogenous progesterone and also enlarged the receptor population. The action did not appear to be an unmasking of cryptic receptors since the effect was not seen in luteal particulates. Elevation of LH receptor numbers by progesterone in immature CL may be a form of intraluteal regulation contributing to the functional maturation of these steroidogenic cells.

Peripheral concentrations of immunoreactive relaxin are generally undetectable during the nonfertile menstrual cycle in humans (1, 2) and rhesus monkeys (3, 4), but become measurable during pregnancy (2, 3, 5–7). The major source of relaxin in primates is the corpus luteum (CL)(5, 8–10). Chorionic gonadotropin (CG) induces relaxin secretion in nonpregnant primates (4,11,12) and thus appears to be a primary stimulus for such induction during early pregnancy. Relaxin is thought to play a role in uterine quiescence during pregnancy (13) and may be an important component for successful pregnancy maintenance.

There is clear evidence for intraluteal production of prostaglandins (PGs) in numerous species and under a variety of experimental conditions. In general, secretion of PGs appears to be elevated in the early corpus luteum (CL) and during the period of luteolysis. Regulation of intraluteal PG production is regulated by a variety of factors. An autoamplification pathway in which PGF-2alpha stimulates intraluteal production of PGF-2alpha has been identified in a number of species. The mechanisms underlying this autoamplification pathway appear to differ by species with expression of Cyclooxygenase-2 (Cox-2) and activity of phospholipase A2 acting as important physiological control points. In addition, a number of other responses that are induced by PGF-2alpha (decreased luteal progesterone, increased endothelin-1, increased cytokines) also have been found to increase intraluteal PGF-2alpha production. Thus, regulation of intraluteal PG production may serve to initiate or amplify physiological signals to the CL and may be important in specific aspects of luteal physiology particularly during luteal regression.

In a preceding study, complete substitution of fish meal protein with cottonseed meal (CM) protein did not affect the survival or growth rate of adult rainbow trout over a 6-mo period. Gossypol, a naturally occurring compound in cottonseeds, has an antifertility effect in terrestrial animals, but information regarding salmonid fish is lacking. Female rainbow trout in this experiment were fed diets with either 0, 25, 50, 75, or 100% (diets 1 to 5) of the fish meal protein replaced with CM protein until first maturation and spawning to study long-term effects on growth and reproduction. Feeding diets containing CM over a total period of 10 mo did not result in differences in growth and mortality compared to the control group (P0.05). Increased CM incorporation levels resulted in decreased (P0.05) blood hemoglobin (10.6 +/- 1.3, 8.4 +/- 1.8, 7.3 +/- 1.1, 6.9 +/- 0.8, and 5.6 +/- 1.4 g/dL) and hematocrit (49.6 +/- 3.9, 38.5 +/- 9.3, 34.4 +/- 3.7, 34.8 +/- 4.9 and 28.0 +/- 6.8%) levels in diets 1 to 5, respectively. The CM incorporation level had no effect (P0.05) on the number of eggs produced per female but led to a reduction (P0.05) in egg weight. Eyed stage survival of embryos was low in all dietary groups and did not show differences (P0.05). However, an increasing CM incorporation level led to a linear increase (P0.05) in the number of females that produced no viable embryos (23.1, 37.5, 42.9, 60.0, and 71.4%). Gossypol in the diet was absorbed by the female trout and transferred to the eggs (0, 2.2 +/- 0.5, 6.7 +/- 1.6, 10.6 +/- 4.2, and 20.0 +/- 2.6 micrograms/g in diets 1 to 5, respectively). A high concentration of gossypol remained in the juveniles at the swim-up stage (endogenous yolk-absorbed) (0.6 +/- 0.3, 2.4 +/- 0.3, 3.4 +/- 0.0, and 4.7 +/- 1.0 micrograms/g, diets 2 to 5, respectively). The findings suggest that replacement of the dietary fish meal protein with CM protein has no effect on fish growth and mortality but may lead to a reduction in reproductive performance in female rainbow trout.

Prostaglandins (PGs) are produced by the corpus luteum (CL) of many domestic and laboratory species and may play a role in CL regulation. The production of PGs by luteal tissue of the rhesus monkey has yet to be clearly elucidated. The production of PGE2, PGF�, and 6-keto-PGF1� by CL from rhesus monkeys and the incubation conditions (time and cell number) that permit assessment of their synthesis were examined. CL (n 3 per characterization) were surgically removed from nonpregnant monkeys during the mid-luteal phase of the menstrual cycle (‘--P8-10 days after ovulation). Luteal tissue was dissociated and the cells were incubated at varying concentrations for increasing periods of time at 37#{176}C. Subsequent to defining incubation conditions, various exogenous factors were examined for their potential to modify PG production. Indomethacin, calcium ionophore, human chorionic gonadotropin (hCG), estradiol-1 7f3 (E2), progesterone (P), testosterone (T), dihydrotestosterone (DHT), and 1-4-6 androstatriene-3, 1 7-dione (ATD) were incubated with luteal cells in increasing doses. PG and P concentrations in the medium were determined by radioimmunoassay. PGs in the medium after 6 h incubation were detectable at all cell concentrations tested (50,000, 100,000, 200,000 cells! tube). Concentrations of PGs and P increased with cell number (p

The rescue of the primate corpus luteum (CL) by CG in early pregnancy is transient, despite a continued rise in circulating CG. To investigate the mechanisms resulting in this response, female rhesus monkeys were given im injections of increasing doses of hCG for 10 days during the menstrual cycle to mimic the pattern of circulating macaque CG observed in early pregnancy. Treatment began 5.3 +/- 0.3 (early luteal phase; n = 4; mean +/- SE), 8.3 +/- 0.5 (midluteal phase; n = 6), or 12.0 +/- 0.4 (late luteal phase; n = 4) days after the midcycle LH surge. Femoral venous blood was collected daily from CG-treated animals from day 8 of the menstrual cycle until the onset of menses. Serum hCG, LH, progesterone (P), and 17 beta-estradiol (E) were measured by RIA. When CG treatment began during the early luteal phase, serum P increased within 9 h after the initial injection (P less than 0.05); elevated concentrations of P persisted through treatment (range, 5.7-10.8 ng/ml) and decreased only after termination of CG injections. When treatment began during the midluteal phase, serum P increased within 9 h (P less than 0.05), peaked the third day of CG treatment (10.4 +/- 2.2 ng/ml), and then declined despite a continued rise in serum CG. Initiation of CG treatment late in the luteal phase also elicited a transient but less profound (peak, 6.4 +/- 0.5 ng/ml) increase in P concentrations. Although P patterns differed among treatment groups (P less than 0.01, by analysis of variance, split plot design), E patterns did not (P greater than 0.05). Serum E increased between 9 and 24 h after onset of treatment and remained elevated until 3-4 days after cessation of CG injections. In summary, treatment that elicited pregnancy-like increases in peripheral CG beginning in the midluteal phase of the menstrual cycle resulted in a transient stimulation of luteal P production similar to that observed in macaques during early pregnancy. This scheme provides a useful model for studying the primate CL during simulated early pregnancy. In contrast, CG administration begun early in the luteal phase resulted in persistent, rather than transient, stimulation of luteal P production. We conclude that the mechanisms involved in the transitory response of the primate CL to circulating CG during pregnancy are inoperative during the early luteal phase of the menstrual cycle.

Several treatment regimens have been used to dissociate bound gonadotropins from their target tissues to quantify numbers of occupied receptors. To compare the efficacy of these methods, we evaluated the ability of a number of treatments to elute bound gonadotropins from corpora lutea of various species. In addition, we examined the capacity of luteal tissue to rebind gonadotro pins after efficacious elution (>90% dissociation of bound gonadotropin). Particulate (20,000 g) preparations of luteal tissue from pseudopregnant rats and from nonpregnant pigs and rhesus monkeys were incubated with '25llabeled human luteinizing hormone (hLH, NIH-LH-I1) or '�51-labeled chorionic gonadotropin (hCG, CR119) for 20 h at 25#{176}C to occupy gonado- tropin receptors. Heat treatment (60#{176} C) eluted >80% of bound '251-hLH from rat tissue within 30 mm, but similar treatment dissociated only 35% ofI-bLH bound to porcine tissue (p 90% of specifically bound "41-bLH from porcine and rat luteal particulates within 5 mm: acetic acid (pH 2.3), fornic acid (pH 2.3), propionic acid (pH 2.3), acetic acid:HC1 (pH 2.3 and 3.3), and MgCI2 (4 M). After 1-2 h at 4#{176}C, exposure to urea (4 M) or acetic acid:HCI (pH 4.3) also eluted> 90% of bound 1251-hLH. Similarly, formic acid (pH 2.3, 5 mm), acetic acid:HCI (pH 2.3 and 3.3, 5 min;pH 4.3, 2 b) dissociated> 95% of bound '251-bCG from monkey luteal particulates. Subsequent to the efficient dissociation of bound hormone from porcine, macaque, and murine luteal tissue, rebinding efficacy differed between treatments (p 90% of the '251-hLH bound by buffer-treated control particulates. Although acetic acid:HC1 (pH 4.3, 2 h) was nearly as effective (>80% rebinding), other treatments that eluted >90% of bound 17�I-hLH markedly suppressed rebinding (24-62% of buffer-treated control uptake). As we observed in using porcine luteal tissue, acetic acid:HC1 (pH 3.3, 5 mm; pH 4.3, 2 h) best preserved the capacity for rebinding to murine and maca que luteal particulates. Protein recovery was not related to rebinding efficiency in any species tested. In conclusion, 1.) the ability of beat treatment (60#{176}C) to dissociate bound gonadotropin from target tissue is species-dependent, 2.) many treatments used to dissociate gonadotropmns from target tissues result in a loss of receptors from the particulate preparation and/or irreversi bly disrupt the ability of membrane receptors to bind gonadotropins, and 3.) estimates of protein recovery cannot account for numbers of receptors disrupted or lost due to the elution treatment. Finally, under optimal conditions, gonadotro pin rebinding following elution remained less than 100% of that of buffer-treated controls. This indicates that the percentage of the receptor population occupied with endogenous gonadotropin was undetectable. As sucb, direct measurement of total receptors (elution :rebinding) lacks the sensitivity to quantify occupied receptors in porcine, macaque, and murine luteal tissue during the ovarian cycle.

Prostaglandin F2α (PGF2α) plays a role in the regression of the corpus luteum (CL) in a number of placental mammals. However, the mechanism of luteal regression has not been extensively studied in marsupials. The objectives of this study were to characterize changes in concentrations of PGF2α within utero-ovarian (UO) tissue/venous plasma during the luteal phase of the estrous cycle in Virginia opossums, to correlate these changes with those of plasma progesterone (P4), and to characterize the peripheral pattern of 13,14-dihydro-15-keto-PGF2α (PGFM) in parturient opossums. Ovaries, uteri, UO venous plasma and peripheral plasma were collected on Days 5, 9 and 12 after induced ovulation (n = 3 to 4 opossums/group). In addition, concentrations of PGFM were measured in peripheral plasma collected from two opossums during late gestation (Days 7,9,11 and 12) and at parturition (Day 13). Concentrations of P4, PGFM and PGF2α in tissue homogenates and plasma samples were estimated by radioimmunoassay. In nonpregnant opossums, peripheral P4 levels were highest on Day 5 (38.8 ± 11.1 ng/ml, x ± SEM) declined on Day 9 (22.6 ± 7.4 ng/ml), and were at basal levels by Day 12 (2.4 ± 0.7 ng/ml). Endometrial concentrations of PGF2α increased (P = 0.056) from Day 5 (15.7 ± 4.1 ng/g) to Day 9 (92.1 ± 61.0 ng/g) and were maintained to Day 12 (97.2 ± 25.7 ng/g). Prostaglandin F2α concentrations in UO plasma increased (P < 0.01) from Day 5 (143.1 ± 32.7 pg/ml) to Day 12 (333.0 ± 32.4 pg/ml). Prostaglandin F2α concentrations in ovarian tissue followed a similar pattern and were correlated with UO concentrations (r = 0.708, P < 0.05). In pregnant opossums, the highest levels of peripheral PGFM were recorded in the peripartum period, when luteal regression would also be expected to occur. The negative temporal relationship between peripheral concentrations of P4 and concentrations of PGF2α in UO tissue/venous plasma observed in this preliminary study is consistent with the notion that PGF2α from the ovary and/or uterus may play a role in CL regression in the opossum.

Two methods for synchronization of parturition in beef cattle were examined. In the first experiment, four groups of cows and heifers were used: untreated (C, n=9), 10 mg flumethasone on day 281 of gestation (F, n=9), 100 mg progesterone daily from days 276 through 283 and 50 mg progesterone on day 284 (P, n=6), and (P+F on day 284, n=7). Variances in gestation lengths (C, 26.01; F, 0.77; P, 11.97; P+F, 1.93) and proportions of cows and heifers with retained placentas (C, 0/9; F, 4/9; P, 1/6; P+E, 0/7) differed significantly among groups. Differences among groups in calving difficulty scores and proportions of dead calves were not significant. Four of the thirteen cows and heifers treated with progesterone required assistance in calving and all four delivered dead calves. Pulling of these calves was not accompanied by uterine contractions. Conversely, the difficult calvings in the control- and flumethasone-treated cows and heifers were accompanied by uterine contractions. In the second experiment, two groups of cows were used: 1) a single injection of 20 mg dexamethasone on either day 276, 277 or 278 followed by injections of saline every 12 h for 2.5 additional days (n=6), and 2) repeated injections of 20 mg dexamethasone every 12 h for 3 days beginning on day 276, 277 or 278 (n=8). The interval from time of first treatment to calving was not different between groups (43.6 and 43.0 h, respectively). Differences between calving difficulty scores, proportions of dead calves and incidence of retained placentas were not significant. Induction very close to the expected calving date could reduce the problems of retained placenta; however, methods must be identified to safely delay parturition.

Prostaglandins (PG) are produced by the corpus luteum (CL) of the rhesus monkey and may be involved in luteal regulation. Intracellular calcium has also been implicated as a mediator of luteolysis in domestic and laboratory species; however, its role in primate luteal function has not been investigated. The objectives of this study were to characterize temporal changes in basal and stimulated luteal PG production by CL of rhesus monkeys, and to examine the effects of calcium ionophore (CaI) on basal and gonadotropin-stimulated progesterone (P) production by the CL. CL were collected at various times after the estimated day of the luteinizing hormone (LH) surge: 5 days (early luteal phase, n = 4), 8-10 days (mid-luteal phase, n = 8), and 12-14 days (late luteal phase, n = 5). Dispersed luteal cells were incubated in the absence and presence of CaI, or with human chorionic gonadotropin (hCG) plus CaI at 37 degrees C for 8 h. PG and P concentrations in the medium were measured by radioimmunoassay. PGE2 and 6-keto-PGF1 alpha production decreased (p less than 0.05) from early luteal phase to mid-luteal phase and remained lower (p less than 0.05) during late luteal phase for all treatment groups. PGF2 alpha production decreased (p less than 0.05) from early to mid-luteal phase and rebounded in late luteal phase to the same level (p greater than 0.05) found in early luteal phase. CaI stimulated (p less than 0.05) basal PG production. The degree of stimulation was similar throughout the luteal phase (p greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of the primate corpus luteum (CL) by endogenous chorionic gonadotropin (CG) in early pregnancy, or by exogenous human (h)CG in simulated early pregnancy, results in a transient elevation of serum progesterone (P) and a persistent elevation of serum 17 beta-estradiol (E). Luteal prostaglandins (PG) may play a role in these responses. The objective of the current study was to correlate luteal PG production and steroidogenic response of CL in vitro with patterns of serum steroids during simulated early pregnancy. CL were removed from rhesus monkeys (n = 26) at 0 h, 9 h, 3 days, 6 days, and 10 days, during prolonged CG exposure of simulated early pregnancy. Dispersed cells were incubated in vitro at 37 degrees C for 8 h. Changes in basal production of P were not significantly correlated with patterns of serum steroids. Maximal stimulation of P production by hCG in vitro (stimulated minus basal) continuously declined (p less than 0.01) from 0 h (means +/- SE, 59.6 +/- 17.9 ng/ml) to 10 days (4.7 +/- 1.8 ng/ml) of simulated early pregnancy. In contrast to patterns of response to hCG, the level of enhancement in P production in response to a maximally stimulatory dose of dibutyryl (db) cyclic adenosine 3',5'-monophosphate (cAMP) declined (p less than 0.05) from 0 h (52.4 +/- 17.6 ng/ml) to 3 days (20.3 +/- 8.4 ng/ml), but was maintained through 10 days (23.7 +/- 11.6 ng/ml) of simulated early pregnancy. As such, desensitization to gonadotropin, which occurred in terms of P production, appears to involve an event subsequent to stimulation of adenylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

The activity of adenylate cyclase was examined in corpora lutea (CL) obtained from rhesus monkeys at specific stages in the luteal phase of the menstrual cycle [3-5, 6-8, 9-12, 13-15, and 16 days (menses) after the midcycle LH surge]. The conversion of [alpha-32P]ATP to [32P]cAMP was used to monitor adenylate cyclase activity. cAMP production in luteal homogenates was assessed in the absence (basal activity) and presence of maximum stimulatory doses of forskolin (100 microM), 5'-guanylylimidodiphosphate [GMP-P(NH)P; 50 microM], GTP (50 microM), and GTP plus increasing doses of hLH and hCG. Basal activity was low in the early luteal phase (days 3-5; mean +/- SE, 1.2 +/- 0.2 pmol cAMP/mg protein X min), increased (P less than 0.05) by the midluteal phase (days 6-8 and 9-12, 2.1 +/- 0.4 and 2.0 +/- 0.3 pmol/mg X min, respectively), and then declined (P less than 0.05) during the late luteal phase (days 13-15 and 16-menses, 1.6 +/- 0.3 and 1.2 +/- 0.5 pmol/mg X min, respectively). Activity stimulated by GTP and GMP-P(NH)P [e.g. GMP-P(NH)P approximately 12 times basal level] followed the same pattern as basal activity during the luteal phase. In contrast, cAMP production in the presence of forskolin did not change significantly throughout the luteal phase. In the midluteal phase (days 6-8 and 9-12; n = 12), hCG and human LH (hLH) stimulated adenylate cyclase in a similar dose-dependent manner. Maximal stimulation of cAMP production by hCG was about 10% greater (P less than 0.05) than that by hLH; the activation constant was 12.3 nM for hCG and 28.3 nM for hLH. The maximal response to hLH and hCG as well as the sensitivity of adenylate cyclase to activation by hLH were greater (P less than 0.05) in the midluteal phase than in the early or late luteal phase. Decreased basal, gonadotropin-stimulated, and guanine nucleotide-stimulated cAMP production and diminished sensitivity of adenylate cyclase to hLH correlated with a decline (P less than 0.05) in circulating progesterone and luteal weight during the late luteal phase. Thus, the adenylate cyclase system of the rhesus monkey CL undergoes significant changes during the luteal phase which are associated with the development and regression of the CL of the menstrual cycle. Mechanisms that modulate gonadotropin and nucleotide activation of adenylate cyclase without interfering directly with the catalytic unit are implicated in the changes that accompany luteolysis.

In women and many nonhuman primates, the life span of the corpus luteum during the nonfertile menstrual cycle is limited to approximately 2 weeks. However, the interval of luteal function is extended when conception and pregnancy follow ovulation. The so-called “rescue” (1) and continued function of the corpus luteum in early pregnancy is essential until the endocrine activities, notably progesterone production, are assumed by the placenta. Ovariectomy or lutectomy prior to the development of placental steroidogenesis causes abortion unless progesterone supplements are provided (2). The luteal-placental shift occurs near the end of the third week of gestation in rhesus monkeys (3) and at 6 weeks in women (2). Hence, the corpus luteum of the fertile cycle must function for an additional 1–4 weeks to ensure continued pregnancy in these species.