Abstract
Ram semen was found to contain 20-hydroxyprostaglandin E1 and 20-hydroxyprostaglandin E2. The relative amounts of the two compounds were almost equal, although ram semen contained at least 10 times more prostaglandin E1 than prostaglandin E2. The accessory genital glands of the ram were analyzed for their capacity to metabolize [14C]arachidonic acid to prostaglandins. Biosynthesis of prostaglandins was only found in microsomes of the mucosa of the ampulla of vas deferens and in microsomes of the vesicular glands. Ram vesicular glands and the ampulla of vas deferens were also found to contain the two 20-hydroxylated E prostaglandins. Microsomes of ram vesicular glands and NADPH metabolized exogenous prostaglandin E2 to 20-hydroxyprostaglandin E2 albeit in low yields. Prostaglandin E2 appeared to be a better substrate than prostaglandin E1. Microsomes of human seminal vesicles and NADPH metabolized exogenous prostaglandin E2 to 19-hydroxyprostaglandin E2. The results show that 19- and 20-hydroxylation of prostaglandins occurs in human and ram seminal vesicles, respectively, and possibly also in the ampulla of vas deferens of the ram. The ram and human enzymes specifically hydroxylated the terminal and the penultimate carbon of prostaglandin E2, respectively.
Highlights
Glandins occurs in humanandram seminal vesicles, respectively, and possibly in the ampulla of vas EXPERIMENTALPROCEDURES deferens of the ram
Prostaglandins in semen are formed by monooxygenases but Ram seminal fluid was pooledin total volumes of 5-10 ml and stored the anatomical locationof the biosynthesis and the naturoef at -80 "C. 19-Hydroxy-PGB1and 19-hydroxy-PGB? were obtained the enzymes have never been determined. 19-Hydroxylated from human seminal fluid (13)
Aftertreatment with alkaliandmethylation,thePGB compounds of ram semen were separated by reversed-phase (i) Microsomes-Microsomes of ram orhuman seminal vesicles HPLC sahsown by Fig. 1.The main prostaglandin were prepared in 0.1 M KHPO, buffer containing 1 mM diclofenac sodium but otherwise as described (13)
Summary
'macology, Karolinska Institutet, Box 60400,104 01 Stockholm, Sweden. The abbreviations used are: PGE, prostaglandin E; A-17-PGEl, 5-10mlof ram semen was added to 10 volumes of acetone and l7,lS-dehydro-PGEl; GC-MS, gas chromatography-mass spectrom- centrifuged or filtered. The acetone extract was evaporated to dryness, etry; HPLC, high performance liquid chromatography; Me3Si, tri- extracted on one or two cartridges of octadecasilane silica as described methylsilyl; Me-, methyl. In some experiments the acetone extract was evaporated and treated with alkali and, after extractive isolation with diethyl ether, the products were methylated and purified by reversed-phase HPLC (system B). He capacity of microsomes from vesicular glands and themucosa of vas deferens to metabolize radiolabeled arachidonic acid was assessed by incubating serial dilutions of the microsomes with the same amount of radioactivity for 3 min a t 37 "C. Ultraviolet Analysis UV spectra were recorded with a Shimadzu 210A spectrophotometer using ethanol as solvent
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