Abstract

Although it is well established that the formation of eicosanoids by ovine intrauterine tissues increases during pregnancy and at the time of labour, the biochemical mechanisms involved remain to be clearly established. In this study, we tested the hypothesis that the gestational and labour-associated increases in eicosanoid formation are associated with a reduction in the activity of the reacylating enzyme, acyl Coenzyme A lysophosphatide acyltransferase (LAT). To evaluate this proposal, in vitro LAT activity was quantified in ovine placenta (cotyledons) obtained during pregnancy (85–147 days of gestation and at the time of labour). Ovine placental LAT increased from 1·81 ± 0·06 nmol/min per mg protein at 85 days of gestation to 2·34 ± 0·10 nmol/min per mg protein at 142 days of gestation (P<0·005, n=15). The apparent K _m did not vary significantly between the 85- and 142-day groups. V_max, however, was significantly greater in the late-gestation group (2·98 ± 0·02 nmol/min per mg protein) than in the mid-gestation group (2·38 ± 0·13 nmol/min per mg protein, P<0·05). In association with labour, placental LAT activity decreased by 16% (1·96 ± 0·13 nmol/min per mg protein) when compared with that observed in tissue obtained from the non-labouring ewe (P<0·01). The data obtained are consistent with the hypothesis that changes in LAT activity in ovine placenta do not contribute to the gestational increase in prostaglandin formation, but a contribution to the labour-associated increase in non-esterified arachidonic acid availability and eicosanoid formation cannot be negated.

Journal of Endocrinology (1996) 148, 241–247

Polychlorinated dibenzo-p-dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been recognized as highly potent developmental and reproductive toxins. We have previously demonstrated effects of TCDD in modulating the expression of rat Sertoli cell secretory products and markers for cell–cell interaction. In this study, we examined the direct biological effects of TCDD in rat Leydig cell primary cultures. Mature rat Leydig cells were purified by Percoll gradient centrifugation and the cell purity was determined by 3β-hydroxysteroid dehydrogenase (3β-HSD) staining and a testosterone induction assay. To examine TCDD-induced biological consequences, we measured the changes in the secretion of progesterone and testosterone, as well as transcript levels of some selected steroidogenic enzymes (i.e. StAR, P450scc, 3β-HSD and CYP17α), in TCDD/human chorionic gonadotropin (hCG) co-treated cells. Our results indicated that TCDD (0.2 or 2 ng/ml) treatment significantly suppressed hCG (5 or 10 ng/ml)-induced testosterone secretion. The suppressive effect aligned with a reduction of progesterone secretion (P<0.05), as well as a decrease of P450scc mRNA and protein expression (P<0.05). The mechanistic action of TCDD was found to be via the reduction of cellular cAMP levels in the hCG-treated cells. This observation was further confirmed, as the TCDD-mediated suppressive effect could be reversed by dibutyryl cAMP co-treatment. The data indicate that TCDD can modulate cAMP signaling in rat Leydig cells to affect the process of steroidogenesis.

ABSTRACT

The capacity of cotyledonary microsomes, prepared from pregnant ewes (20–145 days of gestation), to metabolize exogenous arachidonic acid was quantified using a radiolabel technique. During gestation, the capacity of microsomes to metabolize arachidonic acid increased 25-fold, from 0·36±0·06μmol arachidonic acid/incubation (n = 8) at <100 days of gestation to 9·06±1 ·02μmol arachidonic acid/incubation at 130–145 days of gestation (n = 5; P<0·05). Arachidonic acid was metabolized to prostaglandin E₂ and F_2α, as determined by thin-layer chromatography and reverse-phase high performance liquid chromatography. The profile of prostaglandins synthesized by cotyledonary microsomes did not change throughout gestation. These data suggest that the increase in cotyledonary prostaglandin synthesis that occurs during late gestation and at term may reflect an increase in the tissue content of prostaglandin H₂ synthase.

J. Endocr. (1988) 118, 265–270

ABSTRACT

Inhibition of microsomal prostaglandin (PG) biosynthesis by allantoic fluid, obtained from ewes at 80–120 days of gestation, was examined. Inhibition of cotyledonary microsomal PGE₂ and PGF_2α biosynthesis by lyophilized allantoic fluid occurred in a dosedependent manner. The concentration of allantoic fluid required to inhibit PGE₂ and PGF_2α production by 50% averaged 17·9 ± 3·2 (s.e.m.) mg dry weight/ml (n = 5). Microsomal PG biosynthesis was markedly enhanced by the addition of arachidonic acid (30 μmol/l). Synthesis of PGE₂ and PGF_2α was increased to 245 ± 65% and 184±14% of control (P<0·05, n = 5) respectively. Treatment of cotyledonary microsomes with porcine phospholipase A₂ (PLA₂; 0·125 units/ml) also stimulated PG synthesis, PGE₂ increasing to 216 ± 27% and PGF_2α to 172 ± 14% of control (P<0·05, n=5) respectively. Allantoic fluid (20 mg dry weight/ml) inhibited arachidonic acid-stimulated PG synthesis (PGE₂ by 48·6 ± 13·8% and PGF_2α by 44·2 ± 7·7%) and PLA₂-stimulated PG synthesis (PGE₂ by 60·6±11·6% and PGF_2α by 74·8 ± 8·5%). Allantoic fluid, however, did not affect PLA₂-stimulated release of arachidonic acid from microsomes, thus negating the possibility that allantoic fluid suppresses PG synthesis by inhibiting PLA₂ activity. These data indicate that allantoic fluid inhibits PG production at the level of PG synthase enzymes. Prostaglandin inhibitor(s) in allantoic fluid may play a role in maintaining uterine quiescence throughout gestation and its withdrawal, at term, may be involved in the initiation of labour.

J. Endocr. (1987) 114, 295–300

Abstract

The aim of this study was to determine whether Type II phospholipase A₂ (PLA₂) is released from late pregnant human placental tissue. Placental explants were incubated in vitro and the release of immunoreactive (ir) Type II PLA₂ and PLA₂ enzymatic activity into the medium was determined. Both irType II PLA₂ and PLA₂ enzymatic activity accumulated in the incubation medium in a time-dependent manner (P<0·0001). This release was not associated with a loss of cell membrane integrity, as indicated by measurement of the intracellular enzyme, lactate dehydrogenase, in the incubation medium. The concentration of irType II PLA₂ and PLA₂ enzyme activity present in incubation medium were significantly correlated (P<0·01). Consistent with the hypothesis that Type II PLA₂ may be stored in secretory granules within human placental tissue, incubation in the presence of a membrane depolarising concentration of KCl (60 mm) caused the release of irType II PLA₂ 2·0-fold (P<0·001). PLA₂ enzyme activity released into the incubation medium displays biochemical characteristics consistent with those previously reported for secretory PLA₂ isozymes, that is, a requirement for millimolar concentrations of calcium for optimal enzyme activity, inhibited by reducing agents, such as dithiothreitol and insensitive to heat inactivation. The data obtained in this study establish that irType II PLA₂ is released from term placenta, when incubated in vitro. The release of this extracellularly-active PLA₂ isozyme may contribute to gestational and labour-associated increases in glycerophospholipid metabolism and prostaglandin formation.

Journal of Endocrinology (1997) 153, 151–157

Abstract

Previous studies have demonstrated the existence of several key components of the renin–angiotensin system in the pancreas. In the present study, the localization of angiotensin II receptor subtypes, type I (AT₁) and type II (AT₂), in the mouse and the rat pancreas was studied by immunocytochemistry using specific antipeptide antibodies against the second extracellular loops of AT₁ and AT₂ receptors in conjunction with confocal laser scanning microscopy. In the mouse, immunoreactivity for AT₁ and AT₂ was observed predominantly in the endothelia of the blood vessels and the epithelia of the pancreatic ductal system. Similar distribution of immunoreactivity for AT₁ and AT₂ was also observed. However, the intensity of immunoreactivity for AT₁ and AT₂ was stronger in the rat than that found in the mouse pancreas. Much weaker immunostaining for both AT₁ and AT₂, as compared with that found in ductal regions, was also found in the acini of the rodent pancreas. Together with the previous findings, the present results suggest that AT₁ and/or AT₂ receptors may play a role in regulating pancreatic functions in the rodent.

Journal of Endocrinology (1997) 153, 269–274

Abstract

Parturition in the sheep is preceded by an increase in the synthesis of prostaglandins by intrauterine tissues. Prostaglandin G/H synthase (PGHS) is the central enzyme involved in prostanoid production. Its expression is enhanced during late gestation in the ewe. Recent studies have identified two PGHS isozymes, termed PGHS-1 and PGHS-2. The labour-associated expression of the two isozymes of PGHS in the sheep has not been characterized.

This study investigated the changes in expression of immunoreactive PGHS-1 and PGHS-2 in ovine amnion and placenta following glucocorticoid-induced labour. Ewes underwent surgery to implant fetal and maternal vascular cannulae and uterine electromyogram electrodes between 118 and 125 days of gestation. Fetal sheep were administered either the glucocorticoid betamethasone (n=5) or saline (control n=6) by direct transabdominal intrafetal injection. Ewes from the betamethasone-injected group were killed in the first stage of labour as indicated by uterine electromyographic activity. Ewes from the saline-injected group were killed at the same time to obtain age-matched control tissue. The time taken to euthanasia following induced-labour onset in the glucocorticoid-injected animals was 56·6 ± 0·8 h post-injection.

Plasma endocrine profiles in the maternal and fetal circulation following glucocorticoid injection were comparable to those observed following normal spontaneous delivery. At post-mortem, amnion and cotyledons were collected in liquid N₂ and stored at −70 °C. Solubilized tissue extracts were prepared and analysed by Western blots using polyclonal antibodies to PGHS-1 and PGHS-2 isozymes. Fetal amnion contained PGHS-1 isozyme at day 133 of gestation, as demonstrated in the saline-injected animals. Slightly higher PGHS-1 immunoreactivity was observed following induced-labour onset, although this did not reach statistical significance (P>0·05). PGHS-2 enzyme was not detectable in amnion. PGHS-2 expression was also not induced following labour onset.

In contrast, PGHS-2 demonstrated enhanced expression following glucocorticoid-induced labour in ovine cotyledon. This tissue contained PGHS-1 enzyme, but immunoreactive levels were minimal and demonstrated limited regulation at labour.

These data suggest that the previously reported rise in placental PG production at term in the sheep is predominantly due to increased expression of the PGHS-2 isozyme. This suggests that PGHS-2 contributes to PG production at term labour in sheep or is induced by the mechanisms controlling ovine parturition. PGHS-1 isozyme is produced constitutively in ovine amnion and may contribute to the gestational increase in PG formation by intrauterine tissues.

Journal of Endocrinology (1996) 151, 125–135

Abstract

Sex hormone binding globulin (SHBG) is a homodimeric plasma protein found in mammals that binds sex steroids with high affinity and regulates their bioavailability. The protein is identical in structure and properties to the androgen binding protein (ABP) found in the male reproductive tract. We have isolated a 1245-base pair rabbit SHBG cDNA encoding a reading frame for a signal peptide followed by a protein of 367 amino acids, which shares 79·0, 68·1 and 63·2% amino acid identity with the corresponding human, rat and mouse proteins respectively. Northern blot and hot-nested PCR analyses indicated that rabbit SHBG is produced from a 1·6 kilobase mRNA in the liver of both sexes and in the testis. The rabbit SHBG cDNA was inserted into pGEX-1λT for expression of a glutathione S-transferase/SHBG fusion protein in Escherichia coli. The bacterial product bound 5α-dihydrotestosterone (DHT) in the same manner as the corresponding protein in serum. The dissociation constants (K_d) for rabbit and human SHBGs produced in E. coli were 11·1 ± 1·1 nm and 2·1 ± 0·6 nm respectively, and rabbit SHBG formed a less stable protein-steroid complex (t_½=5 min) than human SHBG (t_½>60 min). Unlike human SHBG, rabbit SHBG does not bind estradiol with high affinity. To aid in the identification of differences in the sequences of rabbit and human SHBG, which determine species differences in steroid-binding affinity and specificity, chimeras containing the 5′-terminal half of SHBG from one species and 3′-terminal half of SHBG from the other species were constructed and expressed. It was found that the chimeric proteins assumed similar steroid-binding affinity and specificity as the wild-type proteins when the amino (N)-terminal half of SHBG was derived from the same species. Replacement of the carboxyl (C)-terminal half of rabbit SHBG by the corresponding region of the human molecule increased the integrity of its steroid-protein complex. This supports the concept that amino acids within the N-terminal half of SHBG constitute the steroid-binding domain while the C-terminal half of the molecule may provide structural stability to the protein and its steroid-binding site.

Journal of Endocrinology (1997) 153, 373–384

Seasonal effects were studied on basal levels of hormones in the serum of adult male Sprague–Dawley rats, which were born and raised under rigorously controlled laboratory conditions. Groups of 90-day-old rats were killed at monthly intervals by rapid decapitation. Significant fluctuations were observed throughout the observation period of 19 months in serum levels of TSH, prolactin, androgens, tri-iodothyronine and LH. Minor fluctuations were observed in serum levels of FSH, corticosterone, parathyroid hormone and thyroxine. The results indicate that male laboratory rats exhibit circannual and semi-annual fluctuations in serum levels of several hormones even though the animals were born, raised and maintained in constant laboratory conditions.

In adult rat testes, blood–testis barrier (BTB) restructuring facilitates the migration of preleptotene spermatocytes from the basal to the adluminal compartment that occurs at stage VIII of the epithelial cycle. Structural proteins at the BTB must utilize an efficient mechanism (e.g. endocytosis) to facilitate its transient ‘opening’. Dynamin II, a large GTPase known to be involved in endocytosis, was shown to be a product of Sertoli and germ cells in the testis. It was also localized to the BTB, as well as the apical ectoplasmic specialization (apical ES), during virtually all stages of the epithelial cycle. By co-immunoprecipitation, dynamin II was shown to associate with occludin, N-cadherin, zonula occludens-1 (ZO-1), β-catenin, junctional adhesion molecule-A, and p130Cas, but not nectin-3. An in vivo model in rats previously characterized for studying adherens junction (AJ) dynamics in the testes by adjudin (formerly called AF-2364, 1-(2,4-dichlorobenzyl)-1H-indazole-3-car-hohydrizide) treatment was used in our studies. At the time of germ cell loss from the seminiferous epithelium as a result of adjudin-induced AJ restructuring without disrupting the BTB integrity, a significant decline in the steady-state dynamin II protein level was detected. This change was associated with a concomitant increase in the levels of two protein complexes at the BTB, namely occludin/ZO-1 and N-cadherin/β-catenin. Interestingly, these changes were also accompanied by a significant increase in the structural interaction of dynamin II with β-catenin and ZO-1. β-Catenin and ZO-1 are adaptors that structurally link the cadherin- and occludin-based protein complexes together at the BTB in an ‘engaged’state to reinforce the barrier function in normal testes. However, β-catenin and ZO-1 were ‘disengaged’ from each other but bound to dynamin II during adjudin-induced AJ restructuring in the testis. The data reported herein suggest that dynamin II may assist the ‘disengagement’ of β-catenin from ZO-1 during BTB restructuring. Thus, this may permit the occludin/ZO-1 complexes to maintain the BTB integrity when the cadherin/catenin complexes are dissociated to facilitate germ cell movement.