Induction of benign prostatic hyperplasia in intact dogs by near-physiological levels of 5 alpha-dihydrotestosterone and 17 beta-estradiol.

Benign prostatic hyperplasia was induced in mongrel dogs treated for 60 days with one silastic implant containing 17 beta-estradiol and four containing 5 alpha-dihydrotestosterone. The condition was characterized by (1) a marked increase of the stromal elements, particularly the stromal septa between the individual glands, (2) a slight increase in prostatic volume, and (3) a morphology that resembled spontaneous complex benign prostatic hyperplasia in the dog. Other groups of animals that remained untreated or received only 17 beta-estradiol or only 5 alpha-dihydrotestosterone did not develop this condition. Prostate volumes decreased by 14% in the estrogen-treated dogs, whereas they increased in the androgen-treated animals by 6% compared to pretreatment prostate volumes. The morphology of the epithelium of the prostates of androgen-treated animals was not different from that of controls despite the increase in prostate volume. The serum 17 beta-estradiol and 5 alpha-dihydrotestosterone concentrations were increased from 25 +/- 2 (mean +/- SEM) and 256 +/- 42 pg/mL, respectively, in control dogs to 52 +/- 37 and 562 +/- 37 pg/mL, respectively, in the dogs treated with the hormone combination. Thus, hormone concentrations were two- to three-fold higher than control values, and the ratio of estradiol-17 beta to 5 alpha-dihydrotestosterone was increased by up to 19%. These data demonstrate that treatment of dogs with low levels of estrogen and androgen may be an excellent model for the study of spontaneous complex benign prostatic hyperplasia in aging men.

Pituitary and Leydig cell function in boars actively immunized against gonadotrophin-releasing hormone.

Crossbred boars were (a) immunized against GnRH conjugated to human serum globulin (200 micrograms GnRH-hSG) in Freund's adjuvant at 12 weeks of age and boosted at weeks 18 and 20 (N = 10), (b) served as controls and received hSG only in adjuvant (N = 10), or castrated at weaning (N = 10). At 24 weeks of age (immediately before slaughter), the boars were challenged with saline or pig LH (1 microgram/10 kg body weight). After slaughter, fresh testicular fragments were incubated with pig LH (0.05 and 0.2 ng/2 ml medium) to assess the effects of immunization on Leydig cell function. Pituitary contents of LH and FSH, and testicular LH receptor content were also measured. The results indicated that plasma LH and testosterone concentrations, pituitary LH content, testicular LH receptor content, testis and sex accessory organ weights were significantly reduced in GnRH-immunized boars compared to hSG-adjuvant controls. However, plasma and pituitary FSH content were not affected by high antibody titres generated against GnRH. The testicular testosterone response to exogenous LH in vivo and in vitro was significantly reduced (P less than 0.05) in GnRH-immunized boars. These results indicate that active immunization against GnRH impairs pituitary and Leydig cell functions in boars.

Changes in testicular morphology in boars actively immunized against gonadotropin hormone-releasing hormone.

Alterations in testicular morphology were studied in boars actively immunized against gonadotropin hormone releasing hormone (GnRH). Ten boars were divided equally into two experimental groups (five GnRH-immunized, and five controls). Antibody production was achieved by conjugating GnRH to human serum globulin (hSG). The GnRH-hSG conjugate was emulsified in complete Freund's adjuvant, and administered to boars at 12 weeks of age. Boars were given a booster in incomplete Freund's adjuvant on week 18 and 20. The presence of high antibody titers to GnRH caused luteinizing hormone and testosterone to decline to nondetectable levels. Morphometric examination showed a reduction in percentage volume in Leydig cells/unit testis, seminiferous tubule diameter and seminiferous epithelial height, and an increase in non-Leydig cell interstitial tissue in GnRH-immunized boars compared with controls. Histologic evaluation displayed severe damage of the seminiferous epithelium, absence of spermatids, incomplete cell associations, disruption of Sertoli cells, formation of multinucleated giant cells, and a striking reduction in size and cytoplasmic structures of Leydig cells in GnRH-immunized animals. These results demonstrate the potent inhibitory effects of GnRH immunoneutralization on the boar reproductive system.

Effect of active immunization against LHRH or LH in boars: reproductive consequences and performance traits.

Forty crossbred boars were equally divided into eight groups at birth. Four groups were immunized (200 micrograms/boar) at 12 wk of age against either luteinizing hormone-releasing hormone (LHRH) conjugated to human serum globulin (LHRH-hSG) in complete Freund's adjuvant (CFA), LHRH-hSG in muramyldipeptide adjuvant (PEP), procine luteinizing hormone (LH) conjugated to hSG (pLH-hSG) in CFA or ovine LH (oLH) in CFA. Equal doses of boosters were given in either PEP or incomplete Freund's adjuvant (IFA) at 16 and 18 wk of age. Two groups of boars were immunized with either hSG + CFA or hSG + PEP (adjuvant controls). Two groups were castrated either at the time of weaning (castrate weaning) or at 16 wk when immunized boars were given their first booster injections (castrate booster). All pigs were slaughtered at 24 wk of age. Serum levels of LH and testosterone (T), LHRH or LH antibody titers, as well as testicular and accessory sex gland weights and histology were determined. By wk 16, LHRH antibody titers began to rise in those boars immunized against LHRH-hSG. Luteinizing hormone-releasing hormone antibody titers on wk 18, 20 and 22 were greater than those at wk 16. By 22 wk of age, LHRH-hSG boars had non-detectable plasma LH and T and reduced weights of testes and acessory sex glands. Boars immunized against oLH did not respond to treatment, whereas pLH-hSG boars showed a reduction in serum T levels and accessory sex gland weights. Immunization had no effect on average daily gain, hot carcass weights or loin eye area.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of gonadotropin secretion in the male: effect of an aromatization inhibitor in estradiol-implanted, orchidectomized dogs.

Testosterone is aromatized to estradiol in both peripheral tissues and the central nervous system. Various authors have suggested that this conversion in the male may be prerequisite for the regulation of gonadotropin secretion by testosterone. Previously, it was reported that inhibition of central nervous system aromatase caused a significant increase in plasma LH in the presence of physiologic testosterone levels (Winter et al, 1983). In order to confirm whether aminoglutethimide, the aromatase inhibitor used in our previous study, either blocked aromatization, or the action of estradiol, the following study was conducted. Fifteen male mongrel dogs were equally divided into three groups. Group 1 dogs were implanted with estradiol-filled polydimethylsiloxane capsules only; Group 2 dogs were implanted with empty capsules and treated with 60 mg b.i.d. of aminoglutethimide; and Group 3 dogs were implanted with polydimethylsiloxane capsules filled with estradiol and treated with aminoglutethimide. Blood samples were drawn for 24 days during pretreatment, capsule implantation, castration, aminoglutethimide administration and capsule removal periods. The postcastration response of both plasma LH and FSH in dogs in groups 1 and 3 was suppressed in the presence of elevated estradiol, whereas that of Group 2 dogs was normal in the absence of estradiol. The results suggest that aminoglutethimide neither directly affects the plasma concentration of either LH or FSH nor blocks the effect of estradiol in inhibiting their release following castration. These data, taken together with our previous work, implicate aromatization of testosterone to estradiol in the control of gonadotropin secretion in the male.

Dose-response studies on male reproductive parameters in dogs with nafarelin acetate, a potent LHRH agonist.

Adult male beagle dogs were administered daily subcutaneous injections of either 0.5 or 2.0 micrograms/kg of a potent LHRH agonist, nafarelin acetate, for 44 days. Although there was a rise in the circulating levels of the gonadotropins and of testosterone following the early injections of agonist, continued treatment caused a marked decline in acute response and basal levels of both LH and testosterone and smaller decreases in the acute FSH response. The decline in LH and testosterone was accompanied by decreases in testicular volume, ejaculated sperm count, sperm motility, ejaculate volume, and duration of ejaculation. The decline in these parameters was more rapid at 2.0 micrograms/kg than at 0.5 micrograms/kg. The profile of responses to 2.0 micrograms/kg could be superimposed on that previously shown for the injection of 10.0 micrograms/kg. At the end of treatment, prostate weights were 36% and 68% of vehicle-treated controls for high- and low-dose animals, respectively. Spermatogenesis was absent in the testes of all agonist-treated animals. Over the dose range tested, the dose-response on all parameters was characterized by a slower evolution to the same maximal effect, rather than by a partial effect. If these data can be extrapolated to man, they would suggest that administration of higher dose levels of LHRH agonists than presently reported should be explored.

Regulation of gonadotropin secretion in the male dog. Role of estradiol.

Steroid regulation of pituitary LH and FSH secretion in the male may be mediated via the direct action of testosterone, or via the conversion of testosterone to either dihydrotestosterone (DHT) or estrogenic steroids. The site of testosterone conversion to these metabolites may be peripherally and/or centrally located. The purpose of this study was to investigate the importance of the conversion of testosterone to estradiol in the control of gonadotropin secretion using the aromatase inhibitor aminoglutethimide (AG). In testosterone-implanted orchidectomized dogs, AG prevented the ability of testosterone to inhibit the post-orchidectomy rise of LH, despite the presence of normal plasma concentrations of both testosterone and estradiol. These data suggest that aromatization of testosterone to estradiol is a necessary step in the regulation of LH secretion. Furthermore, a lack of change in the peripheral concentrations of estradiol in the presence of AG led us indirectly to conclude that inhibition of aromatization occurred centrally rather than peripherally. In the absence of AG, testosterone was unable to maintain intact levels of FSH in the orchidectomized dog for more than six days. Thus, it still remains to be demonstrated whether steroids are of major importance in the regulation of FSH secretion.

Animal model of isolated gonadotropin deficiency. I. Hormonal responses to LHRH immunoneutralization.

Five intact male mongrel dogs, characterized by an episodic secretory pattern of LH and normal serum testosterone concentrations, were actively immunized against LHRH by subcutaneous injections of 200 micrograms of a LHRH-human serum albumin conjugate at 0, 4, and 8 weeks. After 12 weeks, two dogs having the highest antibody titers to LHRH (25% and 51% binding of 125I-LHRH in serum diluted 1:1000 B/Bo) had low to nondetectable serum concentrations of LH and testosterone, whereas serum FSH concentrations were significantly reduced in only one of these dogs. Immunocytochemical techniques showed that the pituitaries of these same two dogs had smaller and fewer LH immunoreactive gonadotropes than did the pituitaries of another three immunized-nonaffected dogs or of the five nonimmunized control dogs. The two LHRH-immunized dogs characterized by hypogonadotropism also had reduced testis (4.0 and 4.0 g) and prostate (2.1 and 1.7 g) weights when compared to control dogs (testis: 12.1 +/- 1.0 g and prostate: 9.2 +/- 1.9 g). LHRH antibody titers in three immunized dogs were demonstrable (8.1, 9.8, and 14.2% B/Bo), but effects on LH, FSH, and testosterone concentrations, pituitary gonadotropes, and reproductive tissue weights were not apparent. The similarity in hormonal and tissue responses observed between dogs effectively immunized against LHRH and men with isolated gonadotropin deficiency suggests that the LHRH-immunized dog may provide a suitable experimental model for the study of patients with isolated gonadotropin deficiency.

Animal model of isolated gonadotropin deficiency. II. Morphologic responses to LHRH immunoneutralization.

Morphologic changes in the male reproductive system of mongrel dogs immunized against LHRH were quantitated using linear measurements and morphometric techniques at the light-microscopic level. Two experimental groups (5 nonimmunized control animals and 5 actively immunized animals) were killed 12 weeks after the primary immunization. No significant differences were observed between three immunized dogs having low LHRH antibody titers (immunized-unaffected) and the five nonimmunized control dogs. The two immunized dogs (affected) with the highest antibody titers against LHRH were characterized by atrophy and dedifferentiation of the testes, prostate, and excurrent ducts. The morphologic changes in the testes of these two dogs were striking and included an apparent arrest or significant reduction in the spermatogenic process, concurrent epithelial degeneration, and apparent diminution of Leydig cell mass. Drastic reductions in the size of the prostatic acini and epithelial cells, as well as loss of secretory granules, reflected depression of function and androgen production. Similarly, in the excurrent ducts decreases in the measured parameters and loss of regional cytoplasmic specialization denoted functional decrescence. This study demonstrates the regressive effects of LHRH immunoneutralization on the morphology of the reproductive system in the male dog and further supports the feasibility of this system as an animal model for the study of isolated gonadotropin deficiency.

Steroid control of gonadotrophin secretion in the orchidectomized dog.

The effects of s.c. administration of oil, testosterone, 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 alpha, 17 beta-diol and oestradiol-17 beta on plasma concentrations of LH and FSH were determined in 5 orchidectomized dogs. The dosages for the androgens and oestradiol-17 beta were 500 and 50 micrograms/kg body weight, respectively. Testosterone and oestradiol-17 beta significantly reduced plasma gonadotrophin concentrations, although the onset and duration of their suppressive effects differed. Dihydrotestosterone and oil had no effect on either gonadotrophin. Administration of androstanediol had no effect on plasma concentrations of LH but did cause a temporary and significant reduction in FSH. It is concluded that testosterone and oestradiol-17 beta are major regulators of gonadotrophin secretion in the male dog, but the 5 alpha-reduction of testosterone seems to play only a minor role in this control.

Evidence that brain aromatization regulates LH secretion in the male dog.

A variety of data suggest an independent role for androgens and estrogens in the regulation of luteinizing hormone (LH) secretion in the male. Estrogens, in the male are primarily derived from testicular androgens that are aromatized both in peripheral tissues and in the CNS. Our prior data suggested a pharmacologic regimen that blocked CNS aromatization without lowering peripheral estrogen or testosterone levels. Such experimental conditions would permit assessment of the relative roles of CNS versus peripheral aromatization in the regulation of LH secretion. We utilized this regimen (aminoglutethimide, a potent aromatase inhibitor, and hydrocortisone) in seven adult male dogs for 14 days. Plasma LH rose to castrate levels, 450% above control values on days 7 and 14. These LH increments stimulated similar rises in androstenedione, testosterone, and dihydrotestosterone. In contrast, plasma estrone and estradiol concentrations remained constant. The induction of castrate LH levels without a concomitant fall in peripheral androgens or estrogens is best explained by a block of central aromatization and thus a reduction in local hypothalamic concentrations. We conclude that aromatization in the CNS rather than peripheral tissues is the more important site with respect to LH negative feedback in the male dog.

Hydrogenated soybean oil (HSO) as a vehicle for the chronic and controlled administration of testosterone in the orchidectomized rat.

The use of a novel vehicle, hydrogenated soybean oil (HSO) for the chronic and controlled release of testosterone (T) in the orchidectomized rat is described. At 60 degrees C, HSO is a liquid in which T is readily soluble. When cooled at approximately 43 degrees C, HSO can be injected into experimental animals without the need of special equipment. At body temperature, the oil forms a solid depot from which T is released over prolonged periods of time. Our studies indicate that a single injection of 5 mg T in HSO in the orchidectomized rat, maintains sex accessory gland weights (AGW) above castrate levels, decreases serum concentrations of LH and maintains serum concentrations of T within the intact range for 10 days after injection. In contrast, these parameters of functional androgenicity are maintained for only three days when T is administered at the same dosage in a conventional oil vehicle. A dose of 15 mg T in HSO maintained serum T concentrations and AGW within intact range for 55 days after injection, but was unable to maintain LH suppression in these castrated animals beyond 14 days after injection.

A noninvasive technique for monitoring changes in canine prostatic size in vivo.

We describe a procedure for monitoring changes in canine prostatic size. Metal beads were sutured to the surface of the prostate and the interbead distances were derived from two X-rays taken at right angles. Prostatic hyperplasia was induced by castration and injection of 5 alpha-androstane-3 alpha, 17 beta-diol and 17 beta-estradiol. Prostatic regression was caused by castration without further treatment. Although growth of the prostate was nonuniform, changes in prostatic volume were shown to be related to the third power of the distance between appropriate beads. This monitoring technique can be repeated every 2nd day if necessary, and thus has an advantage over procedures that require repeated laparotomies. Significant increases in prostatic volume could be detected within 5 days after steroid injection.

Annual variations in plasma levels of testosterone and luteininzing hormone in the laboratory male mongrel dog.

Blood samples, drawn every 15 days (September 1975-September 1976) from four laboratory-housed male mongrel dogs, were assayed by radioimmunoassay for levels of testosterone and LH in the plasma. The mean plasma concentrations of testosterone remained relatively constant for most of the year with the exception of a significant rise in late August and early September. Mean plasma levels of LH showed a cyclic pattern throughout the year which could be represented by a cosine function curve. However, this cyclic pattern of LH was not accompanied by cyclic changes in plasma levels of testosterone and there was no relationship between these two hormones during the period of 1 year. As the cyclic pattern of LH was altered, the plasma level of testosterone began to rise and reached its highest concentration. Since this alteration of the LH cycle occurred before the increased concentrations of testosterone, and since there was no relationship between these two hormones for the period of a year, we have concluded that there may be another hormone(s) involved which either alters the sensitivity of the canine testis to LH or alters the Lh synthesis/release mechanism of the pituitary gland.

Maintenance of physiologic concentrations of plasma testosterone in the castrated male dog, using testosterone-filled polydimethylsiloxane capsules.

Effects of various numbers of polydimethylsiloxane (PDS) capsules filled with testosterone (PDS-T) on plasma testosterone (PT) in castrated male dogs were studied. Dogs were implanted with 1 empty PDS capsule or 1, 3, or 5 PDS-T capsules. Blood samples were collected prior to and after implantation, after castration with capsules in situ, and after capsule removal. The PT was determined in these samples by radioimmunoassay. One empty capsule had no effect on PT concentration; after castration, PT values fell to nondetectable amounts. One PDS-T capsule maintained PT at concentrations above nondetectable amounts after castration, but these concentrations were significantly (P less than 0.05) lower than were preimplantation values. Three or five PDS-T capsules were capable of maintaining PT concentrations in the castrated male dog similar to those concentrations seen in the intact dog.

Plasma concentrations of testosterone and LH in the male dog.

Blood samples were withdrawn every 20 min from 3 conscious intact and 2 castrated mature males during non-consecutive periods of 12 h during the light and dark phases of the lighting schedule (intact dogs) and of 11 h during the light period (castrated dogs). In the intact dogs testosterone concentrations ranged from 0.4 to 6.0 ng/ml over the 24-h period. LH concentrations varied from 0.2 to 12.0 ng/ml. In all animals, LH peaks were clearly followed, after about 50 min, by corresponding testosterone peaks, but no diurnal rhythm could be established. LH concentrations in the castrated dogs were high (9.8 +/- 2.7 (s.e.m.) ng/ml), and still showed an episodic pattern in spite of the undetectable plasma testosterone levels.

Diurnal fluctuations of testosterone and LH in the ram: effect of HCG and gonadotrophin-releasing hormone.

To examine a possible relationship between the release of LH and increasing testosterone levels, blood samples were collected at 2-hr intervals for 24 hr from six mature rams with indwelling jugular cannulae. Although high levels of LH often preceded increases of testosterone, a consistent time relationship between these two hormones was not observed, nor was a diurnal rhythm established for either hormone. The following day, two rams were each injected intravenously with saline, 500 i.u. HCG, or 100 mug synthetic gonadotrophin-releasing hormone (Gn-RH) respectively. Immediately after the injection, blood was collected every 15 min for approximately 4 hr. After Gn-RH, the plasma LH rose 25- to 50-fold within 12 min and was elevated for more than 3 hr. A twofold rise of plasma testosterone followed this increase of LH and testosterone remained high until the end of the experiment. After HCG treatment, the level of testosterone rose threefold within 30 min and did not decline during the experiment, but there was no effect on the LH level. Saline treatment did not influence levels of LH or testosterone. It was concluded that daily fluctuations of testosterone and LH may occur independently, and no pattern is common to all rams. Treatment with Gn-RH results in a rise in both the testosterone and LH levels, but only testosterone is affected by HCG.