ABSTRACT

The aim of this study was to examine the inhibitory effect of the non-aromatizable androgens on FSH-stimulated aromatase activity in porcine granulosa cells. The cells were isolated from medium-sized follicles (4–6 mm) of prepubertal pigs, and cultured under chemically defined conditions in the presence of FSH (1 μg/ml, NIADDK-oFSH-S13) with and without the androgens for an initial 48-h induction period. Subsequently, the spent medium was replaced with fresh medium containing only testosterone as substrate and the cells were reincubated for a further 6 h. The conversion of this steroid to oestradiol-17β during this latter 'test' period was taken as a measure of the aromatase activity. The addition of 5α-dihydrotestosterone (DHT) into cultures of FSH-stimulated cells during the induction period resulted in a definite dose-dependent inhibition (30–70%) of the aromatase activity expressed in the test period. This inhibitory action, of the mixed non-competitive type, is characterized by a decrease in the apparent V _max and an increase in the K _m value, suggestive of an androgen inhibition of FSH-stimulated aromatase synthesis. This inhibition was also shown by the other 5α- and 5β-reduced androgens: 5β-androstanedione was the most effective, while DHT was the least. Other steroids such as pregnenolone and progesterone were inhibitory, but testosterone and diethylstilboestrol were stimulatory. These results suggest an important mechanism for the intrafollicular control of oestrogen synthesis, involving a possible reciprocal relationship between aromatase and 5α-reductase activities.

J. Endocr. (1986) 108, 335–341

The acute effects of pooled porcine follicular fluid (PFF), before and after various methods of processing to eliminate steroids, were studied on the luteinizing hormone releasing hormone (LH-RH)-induced release of FSH and LH by whole pituitary glands, from 34-day-old mice, incubated in vitro for 3–4 h. Charcoal treatment of PFF eliminated the steroids and reduced the inhibitory potency on gonadotrophin secretion. On the other hand, dialysis or ultrafiltration (mol. wt > 10 000) did not reduce the inhibitory activity on gonadotrophin secretion.

Of the three steroids tested, only oestradiol at a concentration of 10⁻¹⁰ mol/l inhibited FSH and LH secretion in vitro. This inhibitory effect was counteracted by the inclusion of the oestrogen antagonist tamoxifen in the incubation medium. The presence of tamoxifen did not decrease the suppression of FSH and LH induced by PFF, suggesting that the inhibition observed under the conditions of incubation was not due to oestrogen. Preincubation of mouse pituitary tissue for 1 h with PFF reduced the subsequent release of bioactive FSH and LH induced by LH-RH. The inhibitory effect of PFF was rapid and sustained. The continuous presence of PFF throughout the incubation period was not necessary for manifestations of the inhibitory effects on gonadotrophin release. The suppression of gonadotrophin secretion was related to the dose of PFF with the curve showing a biphasic pattern. The degree of FSH suppression was uniformly greater than that of LH, showing the preferential nature of the inhibitory effect of PFF. At high doses of PFF, the degree of FSH suppression was decreased significantly. This effect on LH release was less pronounced.

The inhibition caused by PFF in the in-vitro incubation procedure was not due to destruction of LH-RH or the released gonadotrophins.

Adiponectin is an adipocyte-derived hormone that has been implicated recently in the regulation of inflammation in immunocytes, and in lipid metabolism and glucose homeostasis in liver, skeletal muscle and adipocytes. However, information in non-rodent models is limited. We have cloned and sequenced the porcine adiponectin open reading frame and evaluated the regulation of adiponectin in vivo following lipopolysaccharide (LPS) or E. coli administration. The porcine sequence shares approximately 88, 86, 85 and 83% homology with the dog, human, cow and mouse adiponectin respectively, and 79-83% similarity with dog, human, cow and mouse proteins at the amino acid level, based on the translated porcine sequence and GenBank submissions for the other species. Relative serum adiponectin concentrations were not altered in pigs infused with E. coli, and mRNA expression in adipose tissue was not responsive to LPS. However, analysis of serum from very lean vs a substantially fatter genotype of pig indicated that relative circulating adiponectin concentrations are higher (P<0.01) in the lean pigs than in the fatter genotype, and that the difference is established relatively early in the growth curve. Also, incubating pig adipocytes for 6 h with recombinant pig adiponectin resulted in an approximately 30% reduction (P<0.05) in lipogenesis compared with adipocytes under basal conditions and with those incubated in the presence of insulin. This is the first report in any species that adiponectin antagonizes the incorporation of glucose carbon into lipid in the adipocyte, and provides additional evidence that adiponectin acts as an autocrine regulatory factor to regulate energy metabolism.

Abstract

Previous studies have shown a heterogeneous expression of LH receptors in various structures of the porcine ovary. Specially striking was the existence in the preovulatory follicle of inner layers of theca interna cells devoid of LH receptor and the confinement in the corpus luteum of the LH receptor to the external cellular layers. In the present study, we have compared the steroidogenic capabilities of LH receptor-positive and -negative cells using immunocytochemistry for side-chain cleavage P450, 3β-hydroxysteroid-dehydrogenase, 17α-hydroxylase P450 and aromatase P450. We have also examined, using the same methods, the evolution of the various cell types after ovulation and during the development of the corpus luteum.

In preovulatory follicles the inner layers of theca cells which were not labelled with anti-LH receptor antibodies appeared to express the steroidogenic enzymes in a way similar to that of the outer LH receptor-positive cell layers. Ovulation per se did not change the distribution of LH receptors (present in the outer luteal cells and in the granulosa) or of steroidogenic enzymes. However, 48 h after follicular rupture there was a marked decrease in overall labelling with anti-LH receptor antibody, and especially a disappearance of immunostaining in the luteal cells of granulosa origin. In the mid-luteal phase (6 days after ovulation), the receptor content seemed to increase in the peripheral luteal cells derived from the theca but the receptor did not reappear in the granulosa-derived luteal cells. Thus the down-regulation of LH receptor appeared to be reversible in the external thecal layers but irreversible in the granulosa cells. Furthermore, the distribution of the various steroidogenic enzymes in the corpora lutea delineated granulosa-derived from theca-derived cells and showed that only the external layers of the latter expressed the LH receptor.

These results showed the existence in the preovulatory follicle of two theca interna regions expressing the same steroidogenic enzymes but possibly submitted to a different hormonal control. Furthermore, the cells derived from these two regions as well as the cells of granulosa origin showed a distinct pattern of variation of LH receptivity during the development of the corpus luteum.

During these studies we also observed that, in the interstitial tissue, only a minority of cells which derived from remnants of atretic follicles expressed both the LH receptor and the steroidogenic enzymes.

Journal of Endocrinology (1996) 148, 435–446

ABSTRACT

The gonadotrophin preparations that have been used previously to study different aspects of testis function are of limited purity. We have, therefore, purified existing gonadotrophin preparations further. The demonstration of their purity and biological activity are reported as well as their suitability for in-vivo use.

After separation of the subunits of the intact hormones by high-performance liquid chromatography followed by analytical sodium dodecylsulphatepolyacrylamide gel electrolphoresis, no contaminating proteins could be detected in either the FSH or LH preparation. After immunoadsorption, contamination by other pituitary hormones (TSH, prolactin, FSH, LH and GH) was found to be less than 0·002% by weight for all the hormones tested. The biological activity of porcine FSH (pFSH) measured in a Steelman–Pohley assay was 150–170 times more potent than the NIH-FSH-P1 reference preparation. The biopotency of human LH (hLH) in the ovarian ascorbic acid depletion test was measured and appeared to be 8100–8300 IU/mg against the 68/40 International Standard.

The dose-dependent effects of pFSH and hLH on testis weight and the number of FSH and LH receptors were measured in immature (22-day-old) hypophysectomized rats treated for 7 consecutive days. Treatment with FSH induced a dose-dependent increase in testis weight (threefold) when compared with the control. The concentration and total number of LH receptors were increased (two- to sixfold) in a dose-dependent manner. The number of FSH receptors per testis increased while the number of FSH receptors per mg of protein remained unchanged. Administration of human LH to immature hypophysectomized rats had no effect on either LH or FSH receptors, regardless of the dose administered. The plasma concentration of testosterone measured after seven injections of FSH remained at the level of (0·31 nmol/l) found in saline-injected control rats. After 7 days of treatment with hLH, the plasma concentration of testosterone had increased in a dose-dependent manner (three- to tenfold). These results indicate that the gonadotrophin preparations are both highly purified and retained full biological activity and, therefore, are ideal for in-vivo physiological studies.

Journal of Endocrinology (1989) 120, 89–96

The effects of a GH secretagogue, L-692,585 (L-585), and human GH-releasing hormone (hGHRH) on calcium transient and GH release were investigated in isolated porcine pituitary cells using calcium imaging and the reverse hemolytic plaque assay (RHPA). Somatotropes were functionally identified by the application of hGHRH. All cells that responded to hGHRH responded to L-585 application. Perfusion application of 10 microM hGHRH and L-585 for 2 min resulted in an increase in intracellular calcium concentrations ([Ca(2+)](i)) of 53+/-1 nM (mean+/-S.E.M.) (P < 0.01) and 68+/-2 nM (P < 0.01) respectively. The L-585 response was characterized by an initial increase in [Ca(2+)](i) followed by a decline to a plateau level above the baseline. Concurrent calcium imaging with RHPA indicated that the L-585-evoked increase in [Ca(2+)](i) coincided with GH release. L-585 significantly increased the percentage of plaque-forming cells (24+/-3 vs 40+/-6%; P < 0.05) and mean area of plaques (1892+/-177 vs 3641+/-189 micro m(2); P < 0.01) indicating increased GH release. Substance P (SP) analogue ([d -Arg(1),d -Phe(5),d -Trp(7,11)]-SP) blocked, and the hGHRH receptor antagonist ((Phenylac-Tyr(1),d -Arg(2), p-chloro-Phe(6), Homoarg(9), Tyr (Me)(10), Abu(15), Nle(27),d -Arg(28), Homoarg(29))-GRF (1-29) amide) decreased the stimulatory effect of hGHRH. These failed to block the stimulatory effect of L-585, suggesting a different receptor for L-585 from the GHRH receptor. The hGHRH-induced calcium transients and initial peak increase induced by L-585 were significantly decreased by removal of calcium from the bathing medium or the addition of nifedipine, an L-calcium channel blocker. The plateau component of L-585-induced calcium change was abolished by removal of calcium and nifedipine. These results suggest an involvement of calcium channels in GH release. Either SQ-22536, an adenylate cyclase inhibitor, or U73122, a phospholipase C (PLC) inhibitor, blocked the stimulatory effects of hGHRH and L-585 on [Ca(2+)](i) transient, indicating the involvement of adenylate cyclase-cAMP and PLC-inositol triphosphate pathways. These results further suggested that calcium mobilization from internal stores during the first phase of the L-585 response induced an increase in [Ca(2+)](i) whereas calcium influx during the second phase is a consequence of somatotrope depolarization.

Thyroid cell cyclic AMP synthesis is stimulated by β-adrenergic agonists. We have characterized this sensitivity on cultured porcine thyroid cells and have studied its modulation by chronic treatment with thyrotrophin.

The synthesis of cyclic AMP in intact porcine thyroid cells in primary culture was stimulated by the β-adrenergic agonist, isoproterenol. This stimulation was dose-dependent and was inhibited by the β-adrenergic antagonists propranolol and alprenolol. The cell responsiveness (i.e. the response elicited by 5 μm-isoproterenol after 5-min stimulation) was increased when the cells were cultured in the absence of thyrotrophin. Thyrotrophin, when present in the culture medium at the onset of culturing, inhibited this increase. A concentration of 100 μu. thyrotrophin/ml was sufficient to reduce the cyclic AMP response to 15% of its control value. Prostaglandin E₂ or dibutyryl cyclic AMP did not mimic the effect of thyrotrophin. The low sensitivity of thyrotrophin-treated cells to β-adrenergic agonists could be explained by a decreased number of β-adrenergic receptors. [¹²⁵I]Iodohydroxybenzyl pindolol specific binding was ten times greater in membrane preparations of control cells than in membranes derived from thyrotrophin-treated cells. The β-adrenergic sensitivity of cultured thyroid cells was also decreased after long-term treatment by terbutaline. A time- and dose-dependent desensitization was observed.

Thyrotrophin (TSH), cyclic AMP, cyclic GMP and 1-methyl-3-isobutyl-xanthine (MIX) promoted the reassociation of isolated porcine and human thyroid cells into follicular structures in culture and stimulated the uptake of radio-iodide. Monolayer cells were present in all cultures, but in decreasing proportions as the concentration of stimulator was increased. The resting membrane potential of porcine thyroid cells cultured for 4 days in the presence of TSH was −54 ± 3·6 (mean ± s.d.) mV for follicular cells and −31 ± 2·6 mV for monolayer cells. In the absence of TSH, only monolayer cells were present and their membrane potential was −24 ± 2·0 mV. Removal of hormone by washing resulted in hyperpolarization to −70 ± 2·9 mV (follicular cells) or −59 ± 3·4 mV (monolayer cells). Subsequent replacement of TSH, or addition of cyclic AMP, MIX, prostaglandin E₁ (PGE₁) or long-acting thyroid stimulator immunoglobulin resulted in depolarization of previously hyperpolarized cells, to approximately the membrane potential observed before washing. Incubation in MIX resulted in enhanced sensitivity to the depolarizing effect of TSH. Cells cultured in the absence of TSH were unresponsive to TSH or other stimulators. The membrane potential of human thyroid cells behaved similarly in response to TSH, to hormone removal and replacement, and to MIX and PGE1.

ABSTRACT

We have studied the binding of a number of radiolabelled steroids and lipophilic substances to porcine corpus luteum (CL) particulate fractions. Following preincubation of CL homogenates with radiolabelled progesterone or pregnenolone prior to fractionation on continuous sucrose density gradients, a broad peak of binding was observed associated with a particulate fraction of buoyant density 1·05–1·10 g/cm³. Progesterone content also peaked at a similar buoyant density (1·06–1·12 g/cm³). Pretreatment of luteal homogenates with digitonin perturbed the buoyant density of the progesterone-binding particulate fraction to 1·10–1·14 g/cm³ and sharpened the binding peak. Progesterone content was also perturbed to a similar extent by digitonin pretreatment, without release of the steroid. Oestrogens were also sequestered by this fraction, but steroid precursors (cholesterol, cholesterol ester), corticosteroids (cortisol, corticosterone), sterol conjugates (oestrone sulphate, pregnanediol glucuronide) and other lipophilic substances (arachidonic acid, phospholipid, prostaglandins E₁, E₂ and F_2α) were not bound. Androgens were bound weakly by fractions from control gradients but, in the presence of digitonin, significant binding could be demonstrated. Radiolabelled steroids were shown to interact directly with luteal membrane fractions, rather than interacting first with cytosolic steroid receptors which then bound to membranes. Furthermore, [³H]progesterone was not bound by porcine granulosa cell particulate fractions. These observations suggest that this fraction may be involved in sequestration or packaging of progesterone for secretion by the luteal cell.

Journal of Endocrinology (1993) 136, 371–380

ABSTRACT

Thyrotrophin (TSH) and prostaglandin E₂ (PGE₂) increased cellular cyclic AMP (cAMP), calmodulin levels and cAMP phosphodiesterase activity in cultured porcine thyroid cells. Dibutyryl cAMP (dbcAMP), a stable analogue of cAMP, increased calmodulin levels and cAMP phosphodiesterase activity. These results indicate that TSH- and PGE₂-stimulated increases in calmodulin are mediated by cAMP. This increased concentration of calmodulin in turn stimulates cAMP phosphodiesterase. Double reciprocal plots of cAMP hydrolysis yielded two apparent Michaelis constants (K _m); the lower in the 1 μmol/l and the higher in the 10 μmol/l range. Thyrotrophin, PGE₂ and dbcAMP increased the values of maximal velocity without changing the K _m values.

J. Endocr. (1988) 117, 109–114