SUMMARY

The ability of the kidney to extract (inactivate and excrete) argininevasopressin (AVP) from the blood was studied in the isolated perfused rabbit kidney. AVP was added to the blood reservoir to give an initial approximate concentration of 100 μu./ml plasma and samples were taken simultaneously from the arterial and venous side at 5, 15, 30 and 45 min. The AVP concentration in the plasma samples was determined by bioassay in the water-loaded, ethanol-anaesthetized rat. The clearance (extraction ratio x renal plasma flow) of AVP from the blood was concentration-dependent. The average extraction ratio ranged from 0·25 at levels of 100 to 44 μu./ml plasma and 0·42 at levels of 44 to 19 μu./ml plasma. The excretion of AVP in the urine was 23% and 29% respectively of the calculated filtered load, in two isolated perfused kidneys, indicating tubular reabsorption and/or tissue inactivation of the filtered hormone.

*Department of Biochemistry, †Department of Obstetrics and Gynaecology, Program in Reproductive Biology and ‡Department of Pathology, McMaster University, Health Sciences Centre, Hamilton, Ontario, Canada L8S 4J9

(Received 28 May 1974)

Although (−)-trans-Δ⁹-tetrahydrocannabinol (THC), the major psychoactive constituent of marihuana (Mechoulam & Shani, 1970), has a wide variety of pharmacological actions, little is known about the effects of this compound on endocrine function. Nir, Ayalon, Tsafriri, Cordova & Lindner (1973) demonstrated that administration of THC to pro-oestrous rats resulted in suppression of the pre-ovulatory surge of luteinizing hormone. Kolodny, Masters, Kolodner & Toro (1974) have found depressed plasma testosterone levels in marihuana smokers. Gynaecomastia in chronic marihuana smokers has been reported (Harmon & Aliapoulos, 1972) and intimated to result from an effect of THC on prolactin (PRL) secretion. Since oestradiol-17β benzoate (OB) is known to affect PRL release (Chen & Meites, 1970), the present work was undertaken to examine the hypothesis

ABSTRACT

Parturition in the sheep is preceded by an abrupt alteration in placental steroid metabolism causing a shift from progesterone to oestrogen production. This change is believed to be a consequence of the prepartum rise in cortisol in the fetal circulation and involves increases in activities of the enzymes steroid 17α-hydroxylase (cytochrome P-450_17α), steroid C-17,20-lyase, and possibly aromatase and steroid sulphatase. The activity levels have been determined of steroid 17α-hydroxylase, aromatase and steroid sulphatase in placental microsomes in late pregnancy, dexamethasone-induced labour and in natural labour at term. Over the gestational period of 118-140 days, basal levels of placental aromatase were relatively constant (mean value (±s.e.m.) of 5·6 ± 0·5 pmol/min per mg microsomal protein (n = 10)). Pregnenolone and progesterone 17α-hydroxylase activities were undetectable (< 0·5 pmol/min per mg microsomal protein (n = 7)). In six animals in labour induced with infusion of dexamethasone into the fetus, placental aromatase activity increased to a value of 14·0± 1·0 pmol/min per mg protein; placental pregnenolone 17α-hydroxylase, measured in four of the animals, also increased to 453 ± 77 pmol/min per mg microsomal protein. In five animals in natural spontaneous labour with vaginal delivery, aromatase activity was 26·7 ± 5·2 pmol/min per mg microsomal protein and pregnenolone 17α-hydroxylase activity was 141 ±14 pmol/min per mg microsomal protein. Steroid sulphatase activity was barely detectable (< 1·5 pmol/min per mg microsomal protein) during late pregnancy, dexamethasoneinduced labour or natural parturition. Immunoblotting of placental microsomal preparations with a specific antibody to cytochrome P-450 _17α indicated that the glucocorticoid-induced activity of steroid 17α-hydroxylase was associated with an increased content of cytochrome P-450_17α. The cotyledon can then convert pregnenolone to dehydroepiandrosterone and thereby generate aromatizable C₁₉ steroids for the aromatase system to facilitate oestrogen synthesis. Importantly, the ovine placental steroid 17α-hydroxylase inefficiently metabolizes 17α-hydroxyprogesterone to androstenedione and thus progesterone is not a principal precursor of oestrogen. The data are consistent with a mechanism of glucocorticoid action in natural ovine parturition to increase cytochrome P-450_17α synthesis either by stabilizing the mRNA for cytochrome P-450_17α or else acting as a stimulus to transcription of the steroid 17α-hydroxylase gene.

Journal of Endocrinology (1989) 122, 351–359

Abstract

During pregnancy, changes in the IGF axis are associated with changes in maternal metabolism and nutrient repartitioning which are necessary to meet the demands of a growing conceptus. The aim of this study was to assess the IGF axis, maternal weight changes and food intake in female New Zealand White rabbits (n=7) prior to breeding (day 0) and serially throughout pregnancy until term (day 30-31).

The total weight of the pregnant does progressively increased from 4·03±0·06 kg (mean ± s.e.m.) on day 0 to 4·47 ±0·07 kg on day 30 (P<0·001). Maternal tissue mass (total weight minus estimated conceptus weight) increased until day 18, plateaued to day 22/23, and then significantly declined. On day 30, the maternal tissue mass was not significantly different from the non-pregnant value, such that the final increase in total weight was due to conceptus growth. Although the does were fed ad libitum, food intake did not change until day 29 when it decreased to approximately 50% of previous intake (P<0·01).

Maternal serum IGF-I was 499 ± 32 ng/ml on day 0, reached a peak of 832 ± 160 ng/ml on day 21 (P<0·02), and then declined to 341 ± 49 ng/ml on day 30. In contrast, serum IGF-II increased dramatically from a non-pregnant level of 85 ±14 ng/ml to 16 295 ± 2488 ng/ml on day 23 (P<0·001), and then rapidly declined (3335 ± 954 ng/ml, day 30). Changes in serum IGF-binding proteins (IGFBPs) followed a pattern similar to IGF-II, as assessed by Western-ligand blotting. All IGFBPs, especially the 45–40 kDa IGFBP-3 doublet, increased dramatically between days 12 and 24 of pregnancy, and then declined towards term.

In conclusion, we observed unique and dramatic changes in the maternal serum IGF axis that corresponded to periods of maternal weight gain and loss. The tissue source of IGFs and IGFBPs remains undetermined, although it is of note that the time when major changes in the IGF axis were first observed coincided with the time of functional change from yolk sac to placenta in the rabbit.

Journal of Endocrinology (1996) 148, 121–130

Dietary factors play an important role in both the development and prevention of human cancers, including breast carcinoma. One dietary micronutrient, sodium butyrate (NaB), is a major end product of dietary starch and fiber, produced naturally during digestion by anaerobic bacteria in the cecum and colon. NaB is a potent growth inhibitor and initiates cell differentiation for many cell types in vitro. In this study, we investigated the effects of NaB on three human mammary epithelial cells and regulation of the IGF axis, specifically, IGF-binding protein-3 (IGFBP-3), a known growth regulator in human mammary cells, and IGFBP-related protein 2 (IGFBP-rP2)/connective tissue growth factor. NaB inhibited DNA synthesis, as measured by [3H]thymidine incorporation, in estrogen-responsive (MCF-7) and estrogen-non-responsive (Hs578T) breast cancer cells, and normal human mammary epithelial cells (HMEC) to a similar degree (up to 90% inhibition at 1-10 mM concentrations). Treatment of cells with NaB induced histone hyperacetylation, suggesting that NaB exerts its biological effects, at least in part, as a histone deacetylase inhibitor in mammary epithelial cells. Treatment of Hs578T cells with NaB caused an induction of apoptotic cell death. NaB treatment resulted in increased levels of p21(Waf1/Cip1) mRNA and protein in Hs578T cells and distinct upregulation of p27(Kip1) in HMEC, suggesting that NaB activates different genes involved in cell cycle arrest, depending upon the cell type. In the same context, among the IGFBP superfamily members tested, NaB specifically upregulated the expression of IGFBP-3 and IGFBP-rP2. These two proteins are known to be involved in inhibition of mammary epithelial cell replication. Northern blot analysis showed that NaB treatment at 1-10 mM concentrations caused a dose-dependent stimulation of IGFBP-3 mRNA expression in cancerous cells and IGFBP-rP2 mRNA expression in both cancerous and non-cancerous cells. Protein data from Western ligand blot and immunoblot analyses demonstrated parallel results. In summary, we have demonstrated that NaB (i) uniformly suppresses DNA synthesis in both cancerous and non-cancerous mammary cells, and (ii) upregulates IGFBP-3 and IGFBP-rP2 mRNA and protein levels in cancerous and non-cancerous mammary cells. These results provide the first demonstration that butyrate regulates the IGFBP system in the human mammary system.

We examined the effects of three maternal diets (very high fat (VHF), low fat (LF), and control (Purina 5015)) on serum steroids, free fatty acids (FFA), and vaginal pH in National Institutes of Health Swiss mice. Females were fed (VHF, n = 33; LF, n = 33; 5015, n = 48) from 4 to 16 weeks of age. Following breeding, female serum was collected at 0.5 (pre-implantation, early diestrus) or 8.5 (post-implantation, mid-diestrus) days post-coitus (dpc). The serum concentrations of 17β-estradiol, testosterone, progesterone, and FFA were analyzed at both collection points, and vaginal pH at 0.5 dpc. Striking differences in steroids and FFA were observed at 0.5 dpc among the groups. Estradiol was higher in the VHF (14.1 ± 3.0 pg/ml), compared with LF mice (5.2 ± 2.3 pg/ml; P≤ 0.05). In contrast, 0.5 dpc testosterone was lower in the VHF (10.5 ± 3.0 pg/ml) versus the LF group (32.7 ± 8.4 pg/ml; P≤ 0.05). At 8.5 dpc, progesterone was higher in the VHF (89.6 ± 6.7 ng/ml) versus the 5015 group (60.1 ± 4.9 ng/ml; P≤ 0.05). VHF mice had higher FFA concentrations at 0.5 dpc (1.0 ± 0.2 mmol/l) than LF and control mice (0.5 ± 0.1 and 0.6 ± 0.1 mmol/l respectively; P≤ 0.05). At 8.5 dpc, VHF females had higher serum FFA (0.8 ± 0.1 mmol/l) than LF and control females (0.4 ± 0.1 and 0.6 ± 0.1 mmol/l; P≤ 0.05). Mean vaginal pH of VHF females (6.41 ± 0.09) was lower than 5015 females (6.76 ± 0.10; P≤ 0.05). These diet-induced alterations in serum steroid and FFA concentrations might affect several reproductive processes, including preferential fertilization by one class of sperm over the other and sex bias in pre- and post-implantational embryonic development.

Human offspring encounter high amounts of phytoestrogens, such as genistein (GEN), through maternal diet and soy-based formulas. Such chemicals can exert estrogenic activity and thereby disrupt neurobehavioral programming. Besides inducing direct host effects, GEN might cause gut dysbiosis and alter gut metabolites. To determine whether exposure to GEN affects these parameters, California mice (Peromyscus californicus) dams were placed 2 weeks prior to breeding and throughout gestation and lactation on a diet supplemented with GEN (250 mg/kg feed weight) or AIN93G phytoestrogen-free control diet (AIN). At weaning, offspring socio-communicative behaviors, gut microbiota and metabolite profiles were assayed. Exposure of offspring to GEN-induced sex-dependent changes in gut microbiota and metabolites. GEN exposed females were less likely to investigate a novel female mouse when tested in a three-chamber social test. When isolated, GEN males and females exhibited increased latency to elicit their first call, suggestive of reduced motivation to communicate with other individuals. Correlation analyses revealed interactions between GEN-induced microbiome, metabolome and socio-communicative behaviors. Comparison of GEN males with AIN males revealed the fraction of calls above 20 kHz was associated with daidzein, α-tocopherol, Flexispira spp. and Odoribacter spp. Results suggest early GEN exposure disrupts normal socio-communicative behaviors in California mice, which are otherwise evident in these social rodents. Such effects may be due to GEN disruptions on neural programming but might also be attributed to GEN-induced microbiota shifts and resultant changes in gut metabolites. Findings indicate cause for concern that perinatal exposure to GEN may detrimentally affect the offspring microbiome–gut–brain axis.