Mordechai Shemesh

Naturally produced steroid hormones and their release into the environment

Steroidal hormones produced by humans and animals are constantly excreted into the environment in their active forms. The primary steroid hormones are progesterone, estrone, estradiol, testosterone, and cortisol, all of which are lipophilic and poorly soluble in water. The steroids of major concern are estrone and estradiol-17β, since they exert their physiological effects at a lower concentration than other steroids and can be found in the environment in concentrations above their LOEL for fish and plants (10 ng/l). The steroid hormones can be readily measured in run-off, soil, and groundwater, but each steroid has its distinct pathway of transport. Since the major source of steroids in the environment appears to be cattle and chickens, the hormonal steroid input into the environment could be drastically reduced by well-established techniques such as buffer strips and composting.

Granulosa cells as a source and target organ for tumor necrosis factor-α

Tumor necrosis factor (TNF‐α), a 17 kDa cytokine, is a product of activated macrophages which was recently shown to be produced by rat and bovine granulosa cells. In the present work, human granulosa cells derived from preovulatory follicles were used. It was demonstrated that human granulosa cells produce TNF‐α (5–10 units/300 000 cells per 15 h). This production was increased by addition of follicle‐stimulating hormone or by a combination of human chorionic gonadotrophin and CSF to the culture media. TNF was also found in bovine follicular fluid and the concentration was higher in the periovulatory than mid‐cycle follicles. TNF‐α was found to increase prostaglandin F‐2α production by human granulosa cells (P < 0.001). We conclude that granulosa cells are both a source and target organ for TNF‐α.

Levels of Prostaglandin F (PGF) in Bovine Endometrium, Uterine Venous, Ovarian Arterial and Jugular Plasma during the Estrous Cycle

Summary Prostaglandins F (PGF) were measured in uterine vein, ovarian artery, and jugular vein plasma and in the endo-metrial tissues at various times during the bovine estrous cycle, and were compared to peripheral plasma progesterone levels. Four groups of heifers at days 1-5, 10-14, 15-17 and 20-0 of the estrous cycle were studied. Low levels of PGF (48 ± 12 ng/g dry tissue were measured in the endometrium on days 1-14 of the cycle. Higher values (131 ± 9.0) were found at days 15 until the day of estrus (P < 0.001). Similarly, very low levels of PGF were observed in the uterine vein plasma at days 1-14 (0.162 ± 0.044) ng/ml M ± S.E.), whereas on days 15 until the day of estrus the levels ranged from 1.5 to 3.0 ng/ml. The increases in uterine vein PGF on day 15 occured even while peripheral plasma progesterone levels were still high. However, PGF was not elevated in either the ovarian artery or the jugular vein at any time during the cycle, even when uterine vein levels were greatly elevated. No differences in PGF content were detected in endometrial tissue from uterine horns adjacent or opposite to the functional corpus luteum. We thank Dr. J. E. Pike of The Upjohn Company for the gift of prostaglandins used as standards in the radioimmunoassay, and Dr. K. Kirton of The Upjohn Company for supplying PGF antisera. We are grateful to D Lensing for skillful technical assistance.

Use of sperm plasmid DNA lipofection combined with REMI (restriction enzyme-mediated insertion) for production of transgenic chickens expressing eGFP (enhanced green fluorescent protein) or human follicle-stimulating hormone.

Abstract Linearized p-eGFP (plasmid-enhanced green fluorescent protein) or p-hFSH (plasmid human FSH) sequences with the corresponding restriction enzyme were lipofected into sperm genomic DNA. Sperm transfected with p-eGFP were used for artificial insemination in hens, and in 17 out of 19 of the resultant chicks, the exogenous DNA was detected in their lymphocytes as determined by PCR and expressed in tissues as determined by (a) PCR, (b) specific emission of green fluorescence by the eGFP, and (c) Southern blot analysis. A complete homology was found between the Aequorea Victoria eGFP DNA and a 313-bp PCR product of extracted DNA from chick blood cells. Following insemination with sperm lipofected with p-hFSH, transgenic offspring were obtained for two generations as determined by detection of the transgene for human FSH (PCR) and expression of the gene (RT-PCR and quantitative real-time PCR) and the presence of the protein in blood (radioimmunoassay). Data demonstrate that lipofection of plasmid DNA with restriction enzyme is a highly efficient method for the production of transfected sperm to produce transgenic offspring by direct artificial insemination.

Arachidonic Acid and Bovine Corpus Luteum Function

Summary Injections of arachidonic acid directly into the corpora lutea of normal cattle at the 12th or 13th day of the estrous cycle resulted in (a) a decline in jugular vein plasma progesterone levels from an average of 6 ng/ml to 2.5 ng/ml (P < 0.05), (b) an immediate rise in PGF levels in ovarian venous plasma, and (c) a sharp rise in jugular vein plasma estrogen levels. Jugular plasma estrogen peaks were associated with each ovarian vein PGF peak in each animal; jugular plasma LH levels did not change. These results show that the bovine ovary can convert arachidonic acid to PGF and that arachidonic acid administered directly into the corpus luteum results in partial regression of the corpus luteum. PGF appears to stimulate estrogen production by the ovary in the absence of measurable changes of plasma LH. The technical assistance of R. Saatman and Denise Lensing is gratefully acknowledged.

Factors affecting the concentration of testosterone in poultry litter

Abstract Poultry litter used as cattle feed can contain high levels of testosterone (>400 ng/g) and estrogen (>300 ng/g) which can interfere with reproduction. However such high levels of testosterone are not usually found in broiler chicken litter (< 6% of samples). We tested the following parameters to determine what factors could be responsible for highly elevated concentrations of testosterone: sex, age, fermentation. It was found that (1) there is no difference in testosterone content in poultry litter obtained from male (133±12 ng/g) or female broilers (133±13 ng/g); (2) poultry litter from adult roosters have unacceptably high levels of testosterone (670±95 ng/g) and layers have high levels of estrogens (533±50 ng/g); (3) although fermentation of poultry manure generally lowers steroidal hormone content, if antibiotics are added to the incubation, there was a a five‐fold increase in radioimmunoassayable testosterone content (controls 94±6 ng/g vrs treated 578±80 ng/g).

Induction of phytoestrogen production in Medicago sativa leaves by irrigation with sewage water

The phytoestrogenic activity of alfalfa irrigated with sewage water was determined by radioreceptor assay (RRA) and high pressure liquid chromatography (HPLC). It was found that irrigation of alfalfa fields increased the phytoestrogenic activity measured in the leaves from 10 ng estrogen equivalents (est. eqv.) to 137 ng est. eqv. g−1, which was principally due to a rise in coumestrol (<10 μg to 60 μg g−1). Similarly, when plants grown in greenhouses under controlled conditions were irrigated with sewage water, the phytoestrogenic content rose from 34 to 48 est. eqv. g−1; (P<0.01; n = 16). Under hydroponic conditions, addition of 1 nM of estrone to the media resulted in a significant rise in phytoestrogen content in the leaves within 48 hr (from 21.2 to 34.5 est. eqv. g−1; P<0.05, n = 10). Alfalfa irrigated with sewage water may therefore have levels of coumestrol (<30 μg g−1) which can affect fertility in cattle. The agents in the sewage water responsible for increased phytoestrogen content of the alfalfa are probably the steroidal estrogens, estradiol and estrone, which are present in sewage in concentrations ranging from 10 to 300 ng l−1.

Control of bovine placental progesterone synthesis: Roles of cholesterol availability and calcium-activated systems

It was previously reported that dispersed bovine placentome secretes progesterone and that the steroidogenic activity of these cells is stimulated by a calcium-mediated, cyclic nucleotide independent mechanism. In the present study, the influence of substrate availability was explored and the roles of calmodulin and protein kinase C in progestin production examined. Incubation of dispersed fetal cotyledon cells with 25-hydroxycholesterol (25-OH-C), a soluble sterol which readily enters cells and is metabolized to steroid hormones, increased progesterone secretion in a dose-dependent manner. The response to 25-OH-C was dependent on the extracellular calcium concentration. Methyl isobutyl xanthine (MIX) alone also increased pregnenolone as well as progesterone secretion, and the combination of 25-OH-C and MIX stimulated progesterone secretion was inhibited by trifluoperazine. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), caused no major effects on steroidogenesis but the stimulatory effects of MIX or the ionophore A23187 were enhanced in its presence. These findings suggest that (1) basal progesterone secretion by fetal cotyledon cells is limited by cholesterol availability; (2) MIX increases steroidogenesis in part by increasing the synthesis of pregnenolone, but its actions are expressed independently of cholesterol availability; (3) both calmodulin and protein kinase C may participate in the modulation of bovine placental steroidogenesis.

Control of bovine placental progestin synthesis: calcium dependent steroidogenesis is modulated at the site of the cholesterol side chain cleavage enzyme.

We have previously reported that progesterone synthesis in the bovine placenta is regulated by Ca2+ dependent and cyclic nucleotide independent mechanism. In studies conducted to further define the role of Ca2+ in the synthesis of progestins in bovine placental tissue, it was found that both protein kinase C (PKC), as determined by phosphorylation, and cytochrome P-450 side chain cleavage, as determined by Western blot analysis, were detectable in the steroidogenetically active portion of the placentome. To determine the site of action of PKC, fetal cotyledon cells were incubated in media containing 25-hydroxycholesterol in the absence or or presence of 10 ng/ml 12-O-tetradecanoyl-phorbol-13-acetate (TPA). It was found that TPA significantly (P less than 0.05) increased the conversion of the exogenous cholesterol analog to progesterone. To determine if the TPA could act synergistically with calcium activators, fetal cotyledon cells were incubated with either methyl isobutyl xanthine (MIX), an activator of intracellular calcium, or the calcium ionophore, A23187, which increases extracellular calcium influx, or both of these agents, in the presence or absence of TPA. It was found that TPA synergistically increased the conversion of sterol to progestins induced by submaximal concentrations of either MIX or A23187. In the presence of both compounds, TPA induced an even more dramatic increase in progestin synthesis. In experiments in which cyanoketone, an agent that inhibits the conversion of pregnenolone to progesterone, was added, TPA addition resulted in increased pregnenolone production, indicating that side chain cleavage of cholesterol is the site of action. The data, therefore, suggest that: (a) Ca2+ affects mechanisms regulating placental steroidogenesis; (2) one locus of Ca2+ is the cholesterol side chain cleavage reaction; and (3) PKC found in this tissue has a role in the Ca activated progestin production.

Lipoprotein lipase activity in the bovine corpus luteum during the estrous cycle and early pregnancy.

Summary Lipolytic activity measured at pH 8.6 in bovine corpora lutea exhibited classical properties of lipoprotein lipase (LPL) in terms of serum and heparin stimulation and NaCl inhibition. LPL activity was measured in 23 corpora lutea collected at different stages of the estrous cycle and early pregnancy. The LPL activity in cyclic corpora lutea (μmole FA released/hr/100 mg acetone powder) was low at Days 4-8 of the estrous cycle (3.1 ± 1.5: mean ± SE) and at Days 19-20 (1.6 ± 0.6). However, high activity of the enzyme was found at Days 12-15 of the cycle (11.8 ± 1.8); these concentrations were significantly (P < 0.01) elevated over those found at Days 4-8 and 19-20. The enzyme activity began to decline at Days 16-18 of the estrous cycle (5.1 ± 1.7). Low enzyme activity was found in the corpora lutea removed from two cows at Day 22 of pregnancy. Progesterone concentrations were measured in 16 of the 23 corpora lutea and a good correlation (r = 0.75, P < 0.01) was found between lipoprotein lipase and progesterone concentrations of the tissue. The data suggest that LPL may be involved in controlling the transfer of fatty acids, including arachidonic, from plasma lipopro-teins to luteal tissue.