Effects of prostaglandin E and F receptor agonists in vivo on luteal function in ewes.

Loss of progesterone secretion at the end of the estrous cycle is via uterine PGF(2alpha) secretion; however, uterine PGF(2alpha) is not decreased during early pregnancy in ewes to prevent luteolysis. Instead the embryo imparts resistance to PGF(2alpha)-induced luteolysis, which is via the 2-fold increase in prostaglandins E(1) and E(2) (PGE(1), PGE(2); PGE) in the endometrium during early pregnancy. Chronic intrauterine infusion of PGE(1) or PGE(2) prevents spontaneous or an estradiol-17beta, IUD, or PGF(2alpha)-induced luteolysis. Four PGE receptor subtypes (EP(1), EP(2), EP(3), and EP(4)) and an FP receptor specific for PGF(2alpha) have been identified. The objective of this experiment was to determine the effects of EP(1), EP(2), EP(3), or FP receptor agonists in vivo on luteal mRNA for LH receptors, occupied and unoccupied LH receptors, and circulating progesterone in ewes. Ewes received a single treatment of 17-phenyl-tri-Nor-PGE(2) (EP(1), EP(3)), butaprost (EP(2)), 19-(R)-OH-PGE(2) (EP(2)), sulprostone (EP(1), EP(3)), or PGF(2alpha) (FP) receptor agonists into the interstitial tissue of the ovarian vascular pedicle adjacent to the luteal-containing ovary. 17-Phenlyl-tri-Nor-PGE(2) had no effect (P> or =0.05) on any parameter analyzed. Butaprost and 19-(R)-OH-PGE(2) increased (P< or =0.05) mRNA for LH receptors, occupied and unoccupied LH receptors, and circulating progesterone. Both sulprostone and PGF(2alpha) decreased (P< or =0.05) mRNA for LH receptors, occupied and unoccupied LH receptors, and circulating progesterone. It is concluded that both EP(3) and FP receptors may be involved in luteolysis. In addition, EP(2) receptors may mediate prevention of luteolysis via regulation of luteal mRNA for LH receptors to prevent loss of occupied and unoccupied LH receptors and therefore to sustaining luteal function.

Prostaglandin E1 (PGE1), but not prostaglandin E2 (PGE2), alters luteal and endometrial luteinizing hormone (LH) occupied and unoccupied LH receptors and mRNA for LH receptors in ovine luteal tissue to prevent luteolysis.

Loss of luteal progesterone secretion at the end of the ovine estrous cycle is via uterine PGF(2)alpha secretion. However, uterine PGF(2)alpha secretion is not decreased during early pregnancy in ewes. Instead, the embryo imparts a resistance to PGF(2)alpha. Prostaglandins E (PGE; PGE(1)+PGE(2)) are increased in endometrium and uterine venous blood during early pregnancy in ewes to prevent luteolysis. Chronic intrauterine infusion of PGE(1) or PGE(2) prevents spontaneous or IUD, estradiol-17beta, or PGF(2)alpha-induced premature luteolysis in nonbred ewes. The objective was to determine whether chronic intrauterine infusion of PGE(1) or PGE(2) affected mRNA for LH receptors, occupied and unoccupied receptors for LH in luteal and caruncular endometrium, and luteal function. Ewes received Vehicle, PGE(1), or PGE(2) every 4h from days 10 to 16 of the estrous cycle via a cathether installed in the uterine lumen ipsilateral to the luteal-containing ovary. Jugular venous blood was collected daily for analysis of progesterone and uterine venous blood was collected on day-16 for analysis of PGF(2)alpha and PGE. Corpora lutea and caruncular endometrium were collected from day-10 preluteolytic control ewes and day-16 ewes treated with Vehicle, PGE(1) or PGE(2) for analysis of the mRNA for LH receptors and occupied and unoccupied receptors for LH. Luteal weights on day-16 in ewes treated with PGE(1) or PGE(2) and day-10 control ewes were similar (P>or=0.05), but were greater (PPGE(2)>Vehicle-treated ewes. Concentrations of PGF(2)alpha and PGE in uterine venous plasma on day-16 were similar (P>or=0.05) in the three treatment groups. Luteal mRNA for LH receptors and unoccupied and occupied LH receptors were similar (P>or=0.05) in day-10 control ewes and day-16 ewes treated with PGE(2) and were lower (P

Is endothelin-1 luteolytic or antiluteolytic in ewes?

Endothelin-1 (ET-1) has been reported to mediate prostaglandin (PG) F(2)alpha (PGF(2)alpha)-induced luteolysis. Prostaglandins E (PGE; PGE(1)+PGE(2)) are associated with implantation, maternal recognition of pregnancy, and are antiluteolytic and luteotropic in vitro and in vivo. ET-1 increased PGE secretion by bovine luteal tissue in vitro from cows where estrus was not synchronized or when estrus was synchronized with lutalyse and did not affect luteal PGF(2)alpha or progesterone secretion, which does not support the concept that ET-1 is luteolytic or mediates PGF(2)alpha luteolysis. Therefore, the objective of this experiment was to determine whether ET-1 infused every 6h from 2400 h on day 10-1800 h on day 18 of the ovine estrous cycle either into the interstitial tissue of the ovarian vascular pedicle (IP) or intrauterine (IU) adjacent to the luteal-containing ovary was luteolytic in ewes. Treatments were: Vehicle-IP; Vehicle-IU; ET-1-IP; or ET-1-IU. Weights of corpora lutea differed (P< or = 0.05) among treatment groups. Weights of corpora lutea at 1800 h on day 18 were: VEH-IP-247+/-38 mg; VEH-IU-195+/-31 mg; ET-1-IP-626+/-74 mg; and ET-1-IU-542+/-69 mg. Luteal weights on day 18 in ET-1-IP or ET-1-IU-treated ewes did not differ (P> or =0.05), but were heavier (P< or =0.05) than in the Vehicle-IP or Vehicle-IU treatment groups which did not differ (P> or =0.05). Profiles of progesterone in jugular venous plasma of both control groups treated with Vehicle-IP or Vehicle-IU were lower (P< or =0.05) than in ewes treated with ET-1-IP or ET-1-IU, which did not differ (P> or =0.05) between ET-1-IP or ET-1-IU treatment groups. Treatment with ET-1-IP or ET-1-IU increased (P< or =0.05) the PGE:PGF(2)alpha ratio when compared to the Vehicle-IP or Vehicle-IU treatment groups, which did not differ (P> or =0.05) between each other. In summary, ET-1 prevented the decrease in luteal weights and the decline in progesterone, but increased the PGE:PGF(2)alpha ratio when compared to controls. Therefore, it is concluded that ET-1 is not luteolytic in ewes, but instead may be luteotropic or antiluteolytic by altering uterine secretion of the PGE:PGF(2)alpha ratio, since PGE(1) or PGE(2) are luteotropic in vitro and in vivo, PGE(1) or PGE(2) prevent PGF(2)alpha-induced luteolysis in vitro and in vivo, and PGE(1) and PGE(2) increase two-fold in ewe endometrium to prevent luteolysis during early pregnancy.

Mechanism whereby nitric oxide (NO) infused chronically intrauterine in ewes is antiluteolytic rather than being luteolytic.

Nitric oxide (NO) has been reported to be luteolytic in vitro and in vivo in cows. However, an NO donor reversed PGF2alpha-induced inhibition of rat luteal progesterone secretion in vitro and an NO donor or endothelin-1 stimulated bovine luteal tissue secretion of prostaglandins E (PGE; PGE1, PGE2) in vitro without affecting progesterone or PGF2alpha secretion. In addition, chronic infusion of an NO donor into the interstitial tissue of the ovarian vascular pedicle adjacent the luteal-containing ovary prevented the decline in circulating progesterone, while a nitric oxide synthase (NOS) inhibitor did not affect luteolysis. The objective of this experiment was to determine whether an NO donor or NOS inhibitor infused chronically intrauterine adjacent to the luteal-containing ovary during the ovine estrous cycle was luteolytic or antiluteolytic. Ewes were treated either with vehicle (N=5), diethylenetriamine (DETA-control for DETANONOate; N=5), (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETANONOate-long acting NO donor; N=6), l-arginine (N=5), l-nitro-arginine methyl ester (l-NAME-NOS inhibitor; N=6), or NG-monomethyl-l-arginine acetate (l-NMMA; NOS inhibitor; N=5) every 6h from 2400h (0h) on day 8 through 1800h on day 18 of the estrous cycle. Jugular venous blood and inferior vena cava plasma via a saphenous vein cathether 5cm anterior to the juncture of the ovarian vein and inferior vena cava were collected every 6h for analysis for progesterone and PGF2alpha and PGE, respectively, by RIA. Corpora lutea were collected at 1800h on day 18 and weighed. Weights of corpora lutea were heavier (P< or =0.05) in DETANONOate-treated ewes when compared to vehicle, DETA, l-arginine, l-NAME, or l-NMMA-treated ewes, l-arginine luteal weights were heavier than vehicle, DETA, l-arginine, l-NAME, or l-NMMA-treated ewes, and luteal weights of vehicle, DETA, l-NAME, or l-NMMA-treated ewes did not differ amongst each other (P> or =0.05). Profiles of progesterone in jugular venous blood on days 8-18 differed (P< or =0.05) in DETANONOate-treated ewes when compared to vehicle, DETA, l-arginine, l-NMMA or l-NAME-treated ewes, which did not differ (P> or =0.05) amongst each other. The PGE:PGF2alpha ratio profile in inferior vena cava plasma of DETANONOate-treated ewes was increased (P< or =0.05) when compared to all other treatment groups. In a second experiment, conversion of [3H PGE2] to [3H PGF2alpha] by day 15 ovine caruncular endometrium in vitro was determined in vehicle, DETA, or DETANONOate-treatment groups. Conversion of [3H PGE2] to [3H PGF2alpha] was decreased (P< or =0.05) only by DETANONOate. It is concluded that NO is not luteolytic during the ovine estrous cycle, but may instead be antiluteolytic and prevent luteolysis by altering the PGE:PGF2alpha ratio secreted by the uterus.

Is nitric oxide luteolytic or antiluteolytic?

Nitric oxide (NO) has been reported to be luteolytic based on treatment of cows in vivo with an inhibitor of nitric oxide synthase (NOS-produces NO), which delayed the decline in progesterone by two to three days [Jaroszewki J, Hansel, W. Intraluteal administration of a nitric oxide synthase blocker stimulates progesterone, oxytocin secretion and prolongs the life span of the bovine corpus luteum. Proc Soc Exptl Biol Med 2000;224:50-5; Skarzynski D, Jaroszewki J, Bah, M, et al. Administration of nitric oxide synthase inhibitor counteracts prostaglandin F(2alpha)-induced luteolysis in cattle. Biol Reprod 2003;68:1674-81]. The objective of this experiment was to determine the effect of a long acting NO donor or a NOS inhibitor infused chronically into the interstitial tissue of the ovarian vascular pedicle adjacent to the ovary with a corpus luteum on secretion of progesterone during the ovine estrous cycle. Ewes were treated either with Vehicle (N=5); Diethylenetriamine (DETA-control for DETA-NONOate; N=5); (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (DETA-NONOate-long acting NO donor; N=6); or l-nitro-arginine methyl ester (l-NAME-NOS inhibitor; N=6) every 6 h from 24:00 h (0 h) on day 8 through 18:00 h on day 18 of the estrous cycle. Jugular venous blood was collected every 6h for analysis for progesterone and corpora lutea were collected at 18:00 h on day 18 and weighed. Weights of corpora lutea were heavier (P< or =0.05) in DETA-NONOate-treated ewes when compared to Vehicle, DETA, or l-NAME-treated ewes, which did not differ amongst each other (P> or =0.05). Profiles of progesterone in jugular venous blood on days 8-18 differed (P< or =0.05) in DETA-NONOate-treated ewes when compared to Vehicle, DETA, or l-NAME-treated ewes did not differ (P> or =0.05) amongst each other. It is concluded that NO is not luteolytic during the ovine estrous cycle, but may instead be antiluteolytic and prevent luteolysis.

Comparative studies of the toxicity of standard reference materials in various cytotoxicity tests and in vivo implantation tests.

Polyurethane films that contained various amounts of zinc diethyldithiocarbamate (ZDEC) and zinc dibutyldithiocarbamate (ZDBC) were prepared as standard reference materials (SRM). Using three cell lines of V79, L929, and Balb/3T3 cells, the cytotoxicity of the dithiocarbamates and the SRM films were compared by agar diffusion assay, filter diffusion assay, neutral red assay, cell growth assay, and colony assay. Among these in vitro cytotoxicity tests, colony assay was found to be the most sensitive method for detecting the cytotoxicity. The cytotoxic potentials of extracts from SRM films correlated well with the concentrations of ZDEC or ZDBC involved in SRM. When various rubber materials including SRM and surgical rubber latex materials were tested, cytotoxic potentials of these extracts were also correlated with the inflammatory tissue capsule thickness in short-term implantation tests. On the basis of these results, the SRM is judged to be useful for validating test sensitivity, and comparing the correlation between in vitro and in vivo responses.

Comparative studies by cell culture and in vivo implantation test on the toxicity of natural rubber latex materials.

Colony assay using V79 cells, the agar diffusion assay with L929 cells, and the 7-day rabbit muscle implantation test were employed to evaluate the cytotoxicity and tissue toxicity of natural rubber latex (NRL) materials. The in vivo implantation test showed that, among 13 histological parameters, thickness of inflammatory layer was the most useful index to evaluate tissue responses quantitatively. A comparison of the in vivo and in vitro parameters revealed the following correlations between the thickness of the inflammatory layer and cytotoxicity indices: Colony assay of the extracts, IC50: r = 0.80; Agar diffusion assay, Zone index: r = 0.73; Lysis index: r = 0.61. From these results, it appears that the colony assay provides a more reliable prediction of the tissue response than the agar diffusion assay.

Evaluation of biocompatibility, based on quantitative determination of the vascular response induced by material implantation.

The biocompatibility of nine different materials, including positive and negative references, 4 polyurethane based and 3 latex based materials was investigated by (1) cytotoxic assay using V79 chinese hamster cells, (2) the thickness of inflammatory layer at 3 and 7 days after intramuscular implantation test, and (3) the course of the blood flow in tissue reaction around subcutaneously implanted materials using Laser Doppler Flowmetry (LDF) over 14 days following implantation. In addition, for some materials, different modes of sterilization were compared. Although the three methods explore different reactive systems, the material ranking obtained was highly similar for the three methods, suggesting a relative accuracy between them. For one latex however, an absence of cytotoxic effect in culture and a highly intense response by LDF investigation of the same order of magnitude as for the positive reference implant suggest that bioincompatibility may result from the material itself and cannot be exclusively investigated by the leaching of toxic components.

Correlations among chemical constituents, cytotoxicities and tissue responses: in the case of natural rubber latex materials.

Natural rubber latex films obtained from 40 different brands of rubber gloves were tested by quantitative chemical analyses, two cytotoxicity tests (agar-diffusion assay using L929 cells and colony assay using V79 cells) and 7-day implantation test in the rabbit muscle. Multiple regression analysis of these data showed that dithiocarbamate accelerators caused the toxicities whereas antioxidants did not. Thickness of inflammatory layer was the most useful parameter to evaluate tissue response among 13 histological parameters investigated. There were good correlations between the cytotoxicity indices and the thickness of inflammatory layer.