Efficient immunocastration of male piglets by immunoneutralization of GnRH using a new GnRH-like peptide.

Active immunization to immunomodulate regulatory processes suffers from the disadvantage that the antigen is usually 'self' and therefore poorly immunogenic. This has been well illustrated by the long-standing experience with immunocastration vaccines targeting GnRH, a ten amino acid peptide. Not all animals vaccinated with these vaccines are equally affected, even after multiple vaccinations. This is a severe handicap when immunocastration vaccines are applied to male piglets to circumvent surgical castration. Surgical castration is universally practised to prevent boar taint, produced in the testicles of mature boars. Alternative immunocastration is only acceptable if all animals are equally affected using a minimum of vaccinations. Vaccines based on the GnRH peptide itself cannot meet these goals. We showed that using a GnRH-like peptide, a 20 amino acid tandem repeat of the amino acid sequence of the GnRH peptide, these goals can be attained. Using the tandem GnRH peptide to vaccinate male piglets completely abolished the development and endocrinological functioning of the testicles, in contrast to monomer GnRH. These results show that superior antigens can be made for effective immunomodulation by appropriate alteration of the antigen.

Lesions in the hypothalamus after active immunisation against GnRH in the pig.

The terminals of the hypothalamic gonadotrophin hormone-releasing hormone (GnRH) neurons are located within the median eminence and thereby extend beyond the protection of the blood-brain barrier. Thus, these terminals may be subjected to direct autoimmune action in animals that are actively immunised against GnRH. Boars (male pigs) (n = 108) were actively immunised against GnRH by two successive injections with synthetic GnRH, covalently coupled to KLH and dissolved in CFA or IFA. They were killed at 26 weeks of age. Immunised boars were selected on the basis of the resultant testes size, which indicates the effectiveness of the immunisation. The hypothalami of 25 selected animals were studied by histological and immunocytochemical techniques and compared with the hypothalami of three sham- and nine control animals. In the immunised animals, changes in the GnRH system had taken place. These comprised dystrophy of the perikarya and a sharp decrease of the GnRH immunocytochemical reactivity in the terminals within the median eminence. In addition, various degrees of inflammatory reactions were present, particularly within the median eminence. These consisted of tissue disruption by edema, collapse of the capillaries, fibrosis and infiltration with fibroblasts. In addition, accumulations of neurosecretum within the median eminence in combination with hypertrophy of magnocellular neurons within the hypothalamus were present. The reactions were restricted to the median eminence and did not involve other neurohemal organs or other parts of the hypothalamus. A correlation could be established between the incidence of the lesions and the effectiveness of the GnRH autoimmunity (as indicated by the size and endocrine function of the gonads and the anti-GnRH titres). Changes in extra- and intracellular IgG immunocytochemical reactivity within the median eminence indicated the involvement of IgG. The effects were absent from control and sham vaccinated animals and after vaccinations with other compositions of the vaccine. Thus, hypothalamic lesions have been observed in this selected group of animals, vaccinated against GnRH with this particular vaccine.

Rat cholesterol side-chain cleavage cytochrome P-450 (P-450scc) gene. Structure and regulation by cAMP in vitro.

The entire rat P-450scc gene has been cloned, positions/sequences of the exon-intron boundaries (I-IX) are described and 940 base pairs (bp) of 5'-flanking DNA have been sequenced, compared to mouse, bovine and human genes, and analyzed by functional assays. Primer extension analysis mapped the transcription start site 32 bp upstream of the initiator methionine codon. The rat P-450scc promoter was ligated to the human growth hormone (GH) gene yielding p-940RsccGH. This rat P-450scc fusion gene and a mouse gene (p-1500MsccGH) were transiently transfected into primary cultures of rat granulosa cells and Y1 adrenal cells. In the Y1 cells primer extension analysis showed that the rat P-450sccGH gene was expressed at lower basal levels than that of the mouse gene but showed greater stimulation (4-8-fold) in response to 8-bromo-cyclic AMP than the mouse (3-4-fold). Similar results were obtained when the fusion genes were transfected into primary cultures of rat granulosa cells and production of GH was measured in the media of the cells stimulated with forskolin. Furthermore, we document that gonadotropins (follicle-stimulating hormone/luteinizing hormone) can induce luteinization of granulosa cells in vitro, that this process is associated with constitutive maintenance of P-450scc mRNA in the absence of hormones/cAMP, that these events associated with luteinization are differentiation-stage specific and occur only in granulosa cells of preovulatory follicles but not in small antral follicles, that the process can be inhibited by cycloheximide if the protein synthesis inhibitor is present during the first 6 h of exposure to luteinizing hormone but not if added for short durations (3-5 h) later, and that once luteinization is induced P-450scc mRNA expression and progesterone biosynthesis are not strictly dependent on cAMP. Thus, the P-450scc gene is regulated by both cAMP-dependent and cAMP-independent mechanisms, each of which are associated with a specific stage of granulosa cell differentiation. The DNA domains involved in regulating these two diverse processes remain to be determined. Although there was remarkable sequence homology among rat, mouse, bovine, and human genes within 70 bp of the transcriptional start site, no other sequence similarities revealed conserved functional domains among the four genes. Although some cAMP-responsive element-like sequences are present in the rat gene, these were not conserved in the other species; including the mouse which showed high sequence homology with the rat throughout 900 bp of 5'-flanking DNA. Thus, the cAMP domains specific to this and other steroidogenic genes remain to be clearly identified.

Cyclic AMP-dependent and -independent regulation of cholesterol side chain cleavage cytochrome P-450 (P-450scc) in rat ovarian granulosa cells and corpora lutea. cDNA and deduced amino acid sequence of rat P-450scc.

We report the isolating and sequencing of three cDNA clones encoding rat P-450scc, the nucleotide and protein sequences of which are highly homologous to those of bovine and human P-450scc, especially in the putative heme and steroid binding domains. We document that different molecular mechanisms regulate P-450scc in granulosa cells of preovulatory (PO) follicles prior to and after luteinization. Luteinizing hormone/human chorionic gonadotropin (LH/hCG) and cAMP are obligatory to induce P-450scc mRNA in PO granulosa cells in vivo and in vitro. Once P-450scc mRNA is induced as a consequence of the LH/hCG surge it is constitutively maintained by luteinized cells in vivo (0-4 days) and in vitro (0-9 days) in the absence of gonadotropins, is susceptible to modulation by prolactin and is no longer regulated by cAMP. Exposure to elevated concentrations of hCG in vivo for 5-7 h was required for PO granulosa cells to undergo a functional transition establishing the stable luteal cell phenotype. Transient exposure of PO + hCG (7 h) follicles in vitro to the RNA synthesis inhibitor actinomycin D (1 microgram/ml) or the protein synthesis inhibitor cycloheximide (10 micrograms/ml), for 1-5 h prior to culturing the granulosa cells failed to disrupt the induction of P-450scc mRNA, progesterone biosynthesis, and appearance of the luteal cell morphology. Inhibitors of protein kinase A (Rp-cAMPS; 1-500 microM and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8); 1-200 microM) added directly to the luteinized cell cultures also failed to alter P-450scc mRNA in these cells, although the cells contain in vivo amounts of mRNA for RII beta, RI alpha, and C alpha, the primary subunits of protein kinase A found in the rat ovary. These data suggest that expression of the P-450scc gene in rat ovarian follicular cells is regulated in a sequential manner by cAMP-dependent and cAMP-independent mechanisms associated with granulosa cells and luteal cells, respectively.

Aromatase cytochrome P450 and cholesterol side-chain cleavage cytochrome P450 in corpora lutea of pregnant rats: diverse regulation by peptide and steroid hormones.

In previous studies we have shown that aromatase cytochrome P450 (P450arom) mRNA and protein increase markedly in luteal tissue between days 10-19 of gestation, whereas cholesterol side-chain cleavage cytochrome P450 (P450scc) appears to be constitutively maintained regardless of hormonal changes occurring during pregnancy. To identify pituitary and placental hormones that regulate these two P450 enzymes in the rat corpus luteum, serum LH activity and pituitary PRL release were selectively inhibited by administration of LH antiserum (LH-Ab) or CB-154, respectively. Placental hormones were removed by hysterectomy. Hormonal activities were replaced by the administration of hCG, PRL, testosterone (T), or estradiol (E), given individually or in combination. Induction of aromatase mRNA transcripts (3.3, 2.6, and 1.9 kilobases) and protein (54,000 mol wt) between days 10-15 of gestation was blocked by either surgical hysterectomy or LH-Ab treatment. Hysterectomy on day 10 combined with CB-154 abolished not only aromatase mRNA, but also markedly reduced P450scc mRNA (2.0 kilobases) by day 12. Induction of aromatase was partially restored in the day 10-15 hysterectomized rats by treatment with PRL plus E (most effective), PRL plus T, or PRL alone, but not by either T or E alone. Similar results were observed 2 days after hysterectomy (day 12), except that hysterectomy alone caused a transient 3.5-fold increase in P450arom mRNA and protein, most likely due to a transient release of pituitary LH. Aromatase mRNA and protein were also increased in intact pregnant rats treated with hCG between days 10-12. However, no effect of hCG was observed before (days 8-10) or after (days 13-19) midgestation. Likewise, LH-Ab had no effect if given after day 13. Despite hormone-specific regulation of the content of aromatase protein, E biosynthesis in vitro was not strictly related to aromatase enzyme content. We conclude that aromatase mRNA and protein are maintained by PRL at a low level of expression in the first half of pregnancy, can be modulated by LH at midgestation, and are subsequently induced to high levels in the second half of gestation by placental factors (rat placental lactogen-1 and T) and the conversion of T to E in the corpus luteum. P450scc appears to be constitutively maintained. Thus, two P450 genes known to be regulated by LH/cAMP in the rat follicle are controlled by diverse peptide and steroid signal transduction mechanisms in the corpus luteum.

Differential effects of follicle-stimulating hormone, insulin, and insulin-like growth factor I on hexose uptake and lactate production by rat Sertoli cells.

The stimulatory effects of follicle-stimulating hormone (FSH), insulin, and insulin-like growth factor I (IGF-I) on lactate production and hexose uptake by Sertoli cells from immature rats were studied. The time-courses and the maximal stimulatory effects of FSH, insulin, and IGF-I on lactate production were virtually identical. When Sertoli cells were incubated in the presence of FSH in combination with insulin or IGF-I (submaximal doses), additive but no pronounced synergistic effects were observed. The stimulatory effects of FSH and insulin were not dependent on the presence of extracellular calcium. 2-Deoxy-D-glucose (2-DOG), an analogue of D-glucose, was used to investigate the hexose transport system of Sertoli cells. Uptake of 2-DOG was linear in time and virtually all of the intracellular 2-DOG was phosphorylated up to 30 min of incubation; 2-DOG uptake was inhibited by cytochalasin B, but not by cytochalasin E. D-glucose, but not D-galactose, appeared to be an effective competitor of 2-DOG uptake. The Km of 2-DOG uptake was not influenced by FSH, insulin, and IGF-I. FSH had no effect on the Vmax of 2-DOG uptake, whereas insulin and IGF-I caused a 30% stimulation of the Vmax. It is concluded that FSH, insulin, and IGF-I stimulate lactate production by cultured Sertoli cells, but that only insulin and IGF-I stimulate hexose transport. The insulin-like effect of FSH on Sertoli cells may principally involve stimulation of glycolytic enzyme activities.

Insulin-like growth factor I (IGF-I) receptors on Sertoli cells from immature rats and age-dependent testicular binding of IGF-I and insulin.

Insulin-like growth factor I (IGF-I) binding to cultured Sertoli cells from immature rats was quantitatively evaluated. The binding of 125I-IGF-I to the Sertoli cells was specific, time- and pH-dependent and reversible. Scatchard analysis yielded a Kd of 3.5 X 10(-9) M and a binding capacity of 2080 fmol/mg protein. Competition with IGF-I resulted in a half-maximal displacement by 2 nM IGF-I, whereas insulin up to a concentration of 100 nM gave virtually no displacement of IGF-I binding. Similarly, the gonadotropic hormones follitropin and lutropin did not compete with 125I-IGF-I binding. In previous studies, it was shown that cultured Sertoli cells from immature rats bind insulin with a Kd of 1.8 X 10(-9) M and a binding capacity of 8.5 fmol/mg protein. The binding of IGF-I and insulin to a total testis membrane fraction was studied using testes from immature and adult rats. In testis from 21-day-old rats, the maximal specific binding was relatively high for IGF-I (871 +/- 50 fmol/g wet weight) and relatively low for insulin (118 +/- 11 fmol/g wet weight). In adult testis, the maximal specific binding of IGF-I was 324 +/- 40 fmol/g wet weight and that of insulin was 330 +/- 17 fmol/g wet weight. The binding of IGF-I and insulin expressed as fmol bound per testis was increased 6-fold and 45-fold, respectively, between the age of 21 days and adult age. It is discussed that the numbers of receptors for IGF-I and insulin in testis may be developmentally regulated, and that IGF-I may be more important than insulin with respect to testis development and Sertoli cell maturation in the immature rat.

Identification of insulin receptors on rat Sertoli cells.

The binding of insulin to rat Sertoli cells was investigated to establish if effects of insulin on Sertoli cells can be mediated via insulin receptors. Sertoli cells were isolated from the testes of 3-week-old rats, and preincubated for 3 days in the absence of hormones. Binding of 125I-porcine insulin to the Sertoli cells was 75-80% specific and this binding was time- and pH-dependent and reversible. Scatchard analysis of the binding data resulted in curvilinear plots with a high affinity binding of Kd = 1.8 X 10(-9) M. Porcine and bovine insulin competed equally well for 125I-porcine insulin binding. Porcine proinsulin was 10-50 times less potent, corresponding to its lower biological activity. Insulin-like growth factor-I (IGF-I) was 30-40 times less potent, indicating low affinity binding of IGF-I to the insulin receptor. Lutropin which was used as a control gave no competition with the 125I-insulin binding. Affinity labelling of Sertoli cell membrane proteins with 125I-insulin using the cross-linking agent disuccinimidylsuberate revealed binding of insulin to (a) protein(s) of Mr greater than 300,000 or Mr = 130,000 after electrophoresis under non-reducing or reducing conditions, respectively. Affinity labelling with 125I-insulin was largely prevented by unlabelled insulin. It is concluded that the protein of Mr 130,000 may represent the alpha-subunit of the insulin receptor. The presence of insulin receptors as well as IGF-I receptors on cultured rat Sertoli cells may suggest that insulin and IGF-I have specific functions in regulating the maturation and activities of Sertoli cells during the initiation and maintenance of spermatogenesis.

Metabolism of radiolabelled energy-yielding substrates by rat Sertoli cells.

The rates of metabolism in vitro of 3H- or 14C-labelled glucose, pyruvate, glutamine and leucine by Sertoli cells from immature rats were estimated. The overall rate of glucose utilization exceeded by far the rates of oxidation of pyruvate (derived from glucose) via the citric acid cycle and glucose metabolism via the oxidative branch of the pentose phosphate pathway. This pattern of glucose metabolism was not markedly altered after stimulation of glucose metabolism by FSH. The rate of oxidation of exogenous pyruvate indicated that the energy yield from glucose metabolism by Sertoli cells could be dependent on the extracellular concentrations of pyruvate and lactate. There is no evidence that a high rate of aerobic glycolysis is of vital importance for Sertoli cells. In medium containing glucose and all amino acids, 14C-labelled glutamine and leucine were converted to 14CO2 at considerable rates. It was calculated that the oxidation of glutamine and leucine in addition to glucose and fatty acids can yield much of the required energy of Sertoli cells.

Comparison of the effects of insulin and follitropin on glucose metabolism by Sertoli cells from immature rats.

Sertoli cells were isolated from the testes of 3-week-old sterile rats (prenatally irradiated) and incubated for 3 days in the absence of added hormones. Subsequently the effects of follitropin and insulin on glucose metabolism were investigated using this in vitro system. A marked stimulation of net lactate production by either follitropin or insulin was observed within 3 h after addition of the hormones. This response was not inhibited in the presence of the protein synthesis inhibitor cycloheximide. Production of cAMP by the Sertoli cells was markedly enhanced by follitropin, but not at all by insulin. The addition of 0.5 mM dibutyryl cAMP to the incubation medium also resulted in a rapid increase of the rate of lactate production by the Sertoli cells. The stimulation of lactate production by follitropin and insulin was dose-dependent (ED50 of approx. 10 ng NIH-FSH-S13/ml and of approx. 50 ng insulin/ml). It is suggested that the observed effects of insulin on Sertoli cells are mediated via insulin receptors, rather than via receptors for insulin-like growth factors. Within 18 h after addition of either follitropin or insulin the cells became refractory with respect to lactate production to the homologous hormone, whereas the cells could still respond to the heterologous hormone. It is concluded, that follitropin and insulin, acting via different mechanisms, exert similar rapid stimulatory effects on glucose metabolism by Sertoli cells from immature rats in vitro. These effects are not dependent on de novo protein synthesis and may differ from long-term trophic effects of follitropin, insulin, and/or insulin-like growth factors.