Tauroursodeoxycholic acid binds to the G-protein site on light activated rhodopsin.

The heterotrimeric G-protein binding site on G-protein coupled receptors remains relatively unexplored regarding its potential as a new target of therapeutic intervention or as a secondary site of action by the existing drugs. Tauroursodeoxycholic acid bears structural resemblance to several compounds that were previously identified to specifically bind to the light-activated form of the visual receptor rhodopsin and to inhibit its activation of transducin. We show that TUDCA stabilizes the active form of rhodopsin, metarhodopsin II, and does not display the detergent-like effects of common amphiphilic compounds that share the cholesterol scaffold structure, such as deoxycholic acid. Computer docking of TUDCA to the model of light-activated rhodopsin revealed that it interacts using similar mode of binding to the C-terminal domain of transducin alpha subunit. The ring regions of TUDCA made hydrophobic contacts with loop 3 region of rhodopsin, while the tail of TUDCA is exposed to solvent. The results show that TUDCA interacts specifically with rhodopsin, which may contribute to its wide-ranging effects on retina physiology and as a potential therapeutic compound for retina degenerative diseases.

Sertoli cell differentiation in rhesus monkey (Macaca mulatta) is an early event in puberty and precedes attainment of the adult complement of undifferentiated spermatogonia.

In primates, the time course of Sertoli cell proliferation and differentiation during puberty and its relationship with the expansion of undifferentiated type A spermatogonia that occurs at this critical stage of development are poorly defined. Mid and late juvenile and early and late pubertal male rhesus monkeys were studied. Testes were immersion fixed, embedded in paraffin, and sectioned at 5 µm. Sertoli cell number per testis, S-phase labeling (BrdU), and growth fraction (Ki67 labeling) were determined and correlated with corresponding parameters for undifferentiated type A spermatogonia (A dark and A pale). Dual fluorescence labeling was used in addition to histochemistry to monitor spermatogonial differentiation during the peripubertal period using GFRα-1 and cKIT as markers. While the adult complement of Sertoli cells/testis was attained in early pubertal monkeys after only a few weeks of exposure to the elevated gonadotropin secretion characteristic of this developmental stage, the number of undifferentiated type A spermatogonia several months later in mid pubertal monkeys was only 50% of that in adult testes. Both A dark and A pale spermatogonia exhibited high S-phase BrdU labeling at all stages of juvenile and pubertal development. Spermatogonial differentiation, as reflected histochemically and by relative changes in GFRα-1 and cKIT expression, was not observed until after the initiation of puberty. In the rhesus monkey and maybe in other higher primates including human, the pubertal proliferation of undifferentiated spermatogonia is insidious and proceeds in the wake of a surge in Sertoli cell proliferation following termination of the juvenile stage of development.

A re-examination of proliferation and differentiation of type A spermatogonia in the adult rhesus monkey (Macaca mulatta).

BACKGROUND: Companion studies using an experimental non-human primate paradigm known as a testicular clamp indicated that the behavior of undifferentiated type A spermatogonia did not conform fully to earlier classical models. This issue was therefore re-examined in normal monkeys. METHODS: Adult male rhesus monkeys (n = 4) received an i.v. bolus of 5-bromo-2'-deoxyuridine (BrdU): one testis (first) was removed 3 h later and the remaining testis (second) was removed after 11 days and 3 h. Tissue was fixed in Bouin's solution, and numbers of A dark (Ad), small A pale (Aps) and large A pale spermatogonia, differentiating B spermatogonia, S-phase-labeled and degenerating cells were enumerated. Data are given as mean +/- SEM. RESULTS: During the early stages of the seminiferous epithelial cycle in the first testis, Ap spermatogonia (1.3 cells/cross section) were predominantly Aps (nuclear dia., 7.1 +/- 0.1 microm). Aps were never S-phase labeled. Apl (nuclear dia., 8.8 +/- 0.5 microm) appeared in Stages IV-VI and were maximal in Stages VII-X when S-phase labeling of this phenotype at 3 h was greatest. The first generation of B spermatogonia appeared in Stages XI-XII (0.84 cells/cross section). Using cells/cross section, the ratio of Ap (Stages I-V):B1:B2:B3:B4:preleptotene spermatocyte was 1:0.7:1.4:2.8:5.6:11.2. In the second testis, labeled Aps (and Apl) were observed. Ad were not BrdU labeled, and degenerating cells were rarely observed. CONCLUSIONS: The results are not entirely consistent with earlier models of spermatogonial proliferation and differentiation in the monkey. Most notably, our findings suggest that in any one cycle of the seminiferous epithelium only a fraction of Ap spermatogonia is mitotically active.

A selective monotropic elevation of FSH, but not that of LH, amplifies the proliferation and differentiation of spermatogonia in the adult rhesus monkey (Macaca mulatta).

BACKGROUND: Unilateral orchidectomy in monkeys increases spermatogenesis in the remaining testis in association with elevated follicle-stimulating hormone (FSH) secretion and testicular testosterone. The present study examined the relative importance of FSH and testosterone in driving the primate testis toward its spermatogenic ceiling. METHODS: Adult male rhesus monkeys were treated with a gonadotropin-releasing hormone receptor antagonist to inhibit endogenous FSH and luteinizing hormone (LH) secretion. The gonadotrophin drive to the testis was replaced with a pulsatile recombinant human FSH and LH infusion to maintain testicular volume and circulating testosterone and inhibin B at physiological levels. A selective monotropic elevation of FSH or LH that doubled the concentrations of inhibin B or testosterone, respectively, was then imposed for 4 weeks, each in a group of four monkeys. In a third group (n = 4), the gonadotrophin drive remained clamped at physiological levels. Bromo-deoxyuridine was administered 3 h prior to castration, and the effects of the monotropic hormone increments on germ cell number, S-phase labeling and degeneration were determined. RESULTS: Increased FSH, but not LH, produced increases in testicular volume (P < 0.05), the proportion of A pale spermatogonia entering the cell cycle and the numbers of differentiated spermatogonia and more advanced germ cells (P < 0.05). Indexes for spermatogonia labeling and germ cell degeneration were not affected. CONCLUSIONS: Under physiological conditions, circulating concentrations of FSH directly dictate sperm output of the primate testis by regulating the proportion of Ap spermatogonia in the growth fraction. An effect of FSH on survival of the first generation of differentiated B spermatogonia is not excluded.

Structure of complex of synthetic HIV-1 protease with a substrate-based inhibitor at 2.3 A resolution.

The structure of a complex between a peptide inhibitor with the sequence N-acetyl-Thr-Ile-Nle-psi[CH2-NH]-Nle-Gln-Arg.amide (Nle, norleucine) with chemically synthesized HIV-1 (human immunodeficiency virus 1) protease was determined at 2.3 A resolution (R factor of 0.176). Despite the symmetric nature of the unliganded enzyme, the asymmetric inhibitor lies in a single orientation and makes extensive interactions at the interface between the two subunits of the homodimeric protein. Compared with the unliganded enzyme, the protein molecule underwent substantial changes, particularly in an extended region corresponding to the "flaps" (residues 35 to 57 in each chain), where backbone movements as large as 7 A are observed.

The functional significance of FSH in spermatogenesis and the control of its secretion in male primates.

The aim of this review is to provide an integrative analysis of the role of FSH in the control of testicular function in higher primates, including man. Attention is focused on the action of FSH during neonatal development, puberty, and adulthood. Whether FSH is the major determinant of the adult complement of Sertoli cells and whether FSH is obligatory for the initiation, maintenance, and restoration of spermatogenesis is evaluated. The mechanism whereby the circulating concentration of FSH regulates spermatogonial proliferation to dictate the sperm production rate under physiological conditions in the adult is discussed in detail. Inhibin B is the major component of the testicular negative feedback signal governing FSH beta gene expression and FSH secretion, and the evidence for this view is presented. The review concludes with the presentation of a model for the operation of the FSH-inhibin B feedback control system regulating sperm production postpubertally in monkey and man, and with speculation on issues of clinical interest.

Profile of circulating vasoactive substances in hemorrhagic shock and their pharmacologic manipulation.

(a) Hemorrhage in dogs (to 45-50 mm Hg) was associated with a 10-fold increase in plasma renin activity (PRA) which remained elevated throughout the time-course of shock including the irreversible (decompensation) stage. The presence of angiotensin II (AII) in arterial blood was demonstrated by the bloodbathed organ technique and confirmed by blockade with specific AII antagonists (cysteine(8)-AII or isoleucine(8)-AII). The contribution of AII to systemic peripheral resistance during hemorrhage shock in dogs was established by administering AII antagonists which immediately cause a further fall in blood pressure.(b) Plasma catecholamines (CA) steadily increased during hemorrhage and peaked during compensation (a 100-fold increase). The CA decreased progressively during decompensation.(c) Prostaglandin (PG) E-like material was observed in arterial blood for 15-60 min (after hemorrhage); the peak arterial concentration was 2.6 ng/ml blood. Indomethacin (i.v., before 80% of maximum bleedout): (i) confirmed the presence of PGE, (ii) increased blood pressure, and (iii) increased blood loss.(d) Thus: peripheral resistance during hemorrhagic shock seems temporally correlated with blood CA levels (and not PRA), and the renin-AII system contributes to the maintenance of vascular resistance and may markedly decrease perfusion of organs, such as kidney; the administration of the proper combination of specific antagonists of vasoconstrictor humoral substances may radically improve organ perfusion and could contribute to ultimate recovery from hemorrhagic shock.

C-terminally shortened alamethicin on templates: influence of the linkers on conductances.

In order to test the influence of chemical modifications designed to allow covalent coupling of channel-forming peptide motifs into variable sized oligomers, a series of alamethicin derivatives was prepared. The building block encompassing the N-terminal 1-17 residues of alamethicin behaved normally in the conductance assay on planar lipid bilayers, albeit at higher concentration and with a slightly reduced voltage-dependence. A linker Ac-K-OCH(2)C(6)H(4)CH(3)p attached via the epsilon amino group of lysine to the C-terminus of alamethicin(1-17) increased membrane affinity. The latter was further enhanced in a dimer and a tetramer in which alamethicin(1-17) chains were tethered to di- or tetra-lysine linkers, respectively, but macroscopic current-voltage curves displayed much reduced voltage-dependencies and reversed hysteresis. An usual behaviour with high voltage-dependence was restored with the modified dimer of alamethicin(1-17) in which alanine separated the two consecutive lysine residues in the linker. Of special interest was the development of a 'negative resistance' branch in macroscopic current-voltage curves for low concentrations of this dimer with the more flexible linker. Single channel events displayed only one single open state with fast kinetics and whose conductance matches that of the alamethicin heptamer or octamer.

Therapeutic approaches to human immunodeficiency virus: structural studies on G-protein-coupled receptors.

Infection by human immunodeficiency virus (HIV) requires the presence of a chemokine receptor on the susceptible cell. The expression of two different chemokine receptors on macrophages and lymphocytes explains the selectivity of different HIV isolates. The rationale behind the choice of the chemokine receptor (CCR5) expressed on macrophages as a therapeutic target is based on the epidemiological studies of the impact on HIV infectivity of a human mutation that prevents expression of this receptor. CCR5 is a member of the G-protein-coupled receptor family, which has yet to be characterized structurally at atomic resolution. Efforts to model the three-dimensional structure of such receptors and to characterize them experimentally are underway in many laboratories. As an example, structural studies determining the bound conformation of the C-terminal peptide of the alpha-subunit of transducin, the relevant G-protein of vision, with rhodopsin are presented.

Structure of Alamethicin in solution. One- and two-dimensional 1H nuclear magnetic resonance studies at 500 MHz.

We report here the 500 MHz 1H nuclear magnetic resonance spectra of Alamethicin, an icosapeptide antibiotic isolated from Trichoderma viride, in methanol, water and methanol/water mixtures. At this frequency, resonances from all the protons are well-resolved in methanol and may be assigned unambiguously. Spectral assignments were made using two-dimensional spin-echo correlated spectroscopy and by spin-decoupling experiments. The amide coupling constants (JNH-alpha CH) facilitated conformational predictions, which were confirmed in part by two-dimensional nuclear Overhauser experiments. On the basis of these data, we propose a secondary structure for Alamethicin that is alpha-helical toward the N terminus and extended beta-sheet at the C-terminal end. This structure is consistent with earlier circular dichroism measurements (McMullen et al., 1971), infrared attenuated total reflection spectroscopy studies (Fringeli & Fringeli, 1979) and proton exchange data (Davis & Gisin, 1981). The proposed structure is a tightly bound dimer, wherein the beta-sheet is stabilized by intermolecular hydrogen-bonds between opposing molecules. An interesting feature of this structure is that it exhibits both a hydrophobic and a hydrophilic surface. This highly amphiphilic nature of the dimer structure may account for the extensive further aggregation of Alamethicin in water. The 1H n.m.r. spectrum of Alamethicin in water is broad, suggesting extensive association. However, spectral assignments and amide coupling constant measurements in water, which were accomplished by titration of methanolic solution of Alamethicin by water, revealed no gross changes in the basic secondary structure of the molecule.

Testosterone can initiate spermatogenesis in an immature nonhuman primate, Macaca fascicularis.

Four 1-yr-old, i.e. immature, monkeys (M. fascicularis) were treated with testosterone. For the first 21 weeks, they received testosterone capsules sc; thereafter for the remainder of 1 yr, they received weekly injections of 125 mg testosterone enanthate. Four similarly aged monkeys served as untreated controls. The testosterone enanthate injections produced a peak level of 345 +/- 70 nmol/liter (mean +/- SD) after 24 h and the levels were 187 +/- 39 nmol/liter 7 days later. The overall mean level of circulating testosterone in the untreated monkeys was 3.5 +/- 1.1 nmol/liter. Testicular volumes of the treated monkeys became 6 times larger than those of the untreated monkeys. The testicular testosterone concentrations of the treated monkeys were about 2- to 4-fold greater than those of untreated immature monkeys but no more than 82% of the normal adult range. Spermatogenesis in varying degrees of completeness was found in the testes of all four treated monkeys. Moreover, sperm, including some motile cells were found in the ejaculates of two monkeys. These data lead to the conclusion that spermatogenesis can be initiated, although not quantitatively, in an immature nonhuman primate by testosterone treatment.

Follicle-stimulating hormone amplifies the population of differentiated spermatogonia in the hypophysectomized testosterone-replaced adult rhesus monkey (Macaca mulatta).

Although testosterone supports all phases of spermatogenesis in primates, FSH is obligatory for quantitatively normal spermatogenesis. To further investigate the mechanism of action of FSH on spermatogenesis, eight adult male rhesus macaques were hypophysectomized and supplemented daily with cortisone acetate (5 mg/kg BW, sc) and T4 (50 mg/animal, orally). Complete pituitary ablation was established by 1) a decline in mean testicular volume to 8% of the prehypophysectomy value; 2) a failure to secrete gonadotropins in response to 50 micrograms GnRH, iv; and 3) an absence of pituitary in the sella turcica on postmortem examination. Testosterone-filled SILASTIC brand capsules (Dow Corning) were implanted sc to restore testicular testosterone to normal levels. Once the testes had achieved maximum growth under testosterone stimulation alone, the animals were implanted with indwelling venous catheters. In four animals, a pulsatile infusion of human FSH (one pulse of 4 IU/kg BW every 3 h) was administered for 12 days, and the other four monkeys received vehicle. Testosterone replacement continued throughout the experiment. At the termination of the 12 days of FSH stimulation or vehicle administration, the right testes were removed. The left testes were removed 22 days later to investigate whether testosterone was able to maintain the effects, if any, of FSH stimulation. Portions of each testis were fixed in Bouin's solution and subsequently prepared for histological examination, whereas other portions were frozen in liquid nitrogen for determination of testicular testosterone content. Five hundred cross-sections of seminiferous tubules in periodic acid-Schiff-hematoxylin-stained histological sections were randomly selected from each testis. The stage of the seminiferous epithelial cycle in these sections was identified, and the germ cells and Sertoli cells were counted in each. All cell counts were corrected by the Abercrombie method and expressed per cross-section of seminiferous tubule. Treatment with FSH for 12 days failed to influence the numbers of either Sertoli cells or Ad and Ap stem spermatogonia. In striking contrast, the number of all four generations of differentiated (B1, B2, B3, and B4) spermatogonia were significantly amplified by stimulation with human FSH for 12 days. As reflected by the analysis of the left testes collected 22 days after termination of the gonadotropin treatment, the progeny of these B spermatogonia were not maintained in the absence of FSH. In conclusion, the results of the present study indicate that the action of FSH to quantitatively maintain spermatogenesis in the rhesus monkey is mediated by a selective amplification of B spermatogonia.

Dynamics of the follicle-stimulating hormone (FSH)-inhibin B feedback loop and its role in regulating spermatogenesis in the adult male rhesus monkey (Macaca mulatta) as revealed by unilateral orchidectomy.

The purpose of this study was to document the morphological changes in the seminiferous epithelium that underlie the compensatory testicular hypertrophy observed in response to unilateral orchidectomy (UO) in the adult rhesus monkey and to describe the concomitant response in the endocrine feedback loops controlling testicular function in this species. Adult male monkeys were implanted with indwelling venous catheters; seven animals were then subjected to UO (data are presented from six) and three to sham UO. Profiles of circulating concentrations of FSH, LH, testosterone (T), inhibin B, and pro-alpha-C were monitored in 12-h series of sequential blood samples collected before, on the day of UO (day 0), and on days 1, 2, 4, 8, 16, 32, and 42 or 43 after UO. In the UO monkeys, the remaining testis was taken on day 44. Sertoli and germ cells in the removed and remaining testes were counted and expressed either as number per testis or, in the case of the differentiated spermatogonia (B1, B2, B3, and B4), as number per cross-section of the seminiferous tubule. UO was associated with a marked increase in the number of all germ cells more mature than undifferentiated spermatogonia (Ap) in the remaining testis. Sertoli cell number, on the other hand, did not change, and it is therefore reasonable to propose that the primary locus of the spermatogenic compensation was the differentiated spermatogonia. The additional finding that the relationship between the number of Sertoli cells and total germ cells in the remaining testis became robust (r = 0.92; P < 0.01 vs. r = 0.44; P > 0.05 for the removed testis) indicated that in the monkey, spermatogenesis does not normally operate at its ceiling. The increased drive to the seminiferous tubule of the remaining testis is hypothesized to be mediated by the sustained increase in FSH secretion that was observed after UO, although a role for increased testicular T production cannot be excluded. The stimulus for increased FSH secretion was presumably provided by the abrupt, 50% decline in circulating inhibin B levels. Interestingly, inhibin B secretion by the remaining testis was not dramatically affected by UO, and therefore, the deficit in circulating levels of this hormone and thus the error signal to FSH secretion were maintained for the duration of the experiment. In contrast, the changes in circulating LH and T concentrations were only transient, and within 48 h of UO, these hormonal parameters had returned to control values. The mechanisms by which the remaining testis rapidly acquires the capacity to double T production in the face of an unchanging LH drive remains to be determined. The foregoing body of evidence suggests that sperm output by the monkey testis is regulated by the circulating concentration of FSH and that in physiological situations, FSH secretion is insufficient to stimulate spermatogenesis to its ceiling. The results also indicate that FSH secretion is controlled by a feedback system in which the feedforward arm (FSH-inhibin B) is less robust than the feedback loop (inhibin B-FSH). Thus, a decrease in the inhibin B feedback signal results in a sustained increase in FSH secretion that drives the testes toward their spermatogenic ceiling, which is presumably set by Sertoli cell number.

Circulating inhibin alpha concentrations in infant, prepubertal, and adult male rhesus monkeys (Macaca mulatta) and in juvenile males during premature initiation of puberty with pulsatile gonadotropin-releasing hormone treatment.

Circulating inhibin alpha concentrations were determined in infant, juvenile, and adult male rhesus monkeys with a RIA employing antisera to a synthetic fragment of the alpha-subunit of porcine inhibin. Binding of tracer, [DSer1,Nle5]human inhibin alpha(1-25)-Gly-125I-Tyr, to antibody was inhibited by standard, [DSer1,Nle5]human inhibin alpha(1-25)-Gly-Tyr. and by plasma from adult male monkeys in a parallel fashion. Castration in adults resulted in a 5-fold decline in the levels of immunoreactivity in plasma. Mean (+/- SE) plasma inhibin alpha concentrations in infants and adults (322.9 +/- 51.9 and 460.1 +/- 43.9 pg/ml, respectively) were significantly higher (P less than 0.05) than those in juveniles (191.3 +/- 28.3 pg/ml). Moreover, initiation of puberty in juvenile males, 13-18 months of age, with a chronic (10- to 12-week) intermittent iv infusion of GnRH (0.1 microgram/min for 3 min every 3 h) resulted in a progressive rise in circulating inhibin alpha that plateaued, after 5 weeks of pituitary stimulation, at concentrations (343.9 +/- 38.2 pg/ml) comparable to those of infants and adults and twice those observed before initiation of the pulsatile infusion of GnRH. Circulating FSH concentrations increased during the first week of GnRH stimulation from 2.7 +/- 0.1 ng/ml before treatment to 6.0 +/- 1.2 ng/ml, where they remained for the duration of the experiment. Testosterone secretion during the initiation of precocious puberty occurred in discrete episodes that were robustly correlated with GnRH-induced LH discharges. In contrast, changes in circulating inhibin alpha concentrations over the 3-h interval between GnRH pulses were unremarkable. Activation of Sertoli and Leydig cells during initiation of puberty in the juvenile males, as reflected by circulating inhibin alpha and testosterone concentrations, respectively, occurred with similar time courses. At the time of orchidectomy, 10-12 weeks after initiation of GnRH treatment, testicular tissue was prepared for histological examination. In spite of a 2-fold gain in testicular weight and in hypertrophy of Sertoli cells in association with GnRH stimulation, maturation of the germinal epithelium did not progress past prophase I spermatocytes, and the number of these latter cells was meager. These findings indicate that the testis of the infant primate, like that of the adult, secretes significant amounts of inhibin, and that the quiescent Sertoli cell of the juvenile males may be readily provoked by appropriate gonadotropin stimulation into producing inhibin. The results also fail to provide evidence for the view that changes in circulating inhibin concentrations are robustly related, in an inve

Effects of orchidectomy on gonadotropin and inhibin subunit messenger ribonucleic acids in the pituitary of the rhesus monkey (Macaca mulatta).

Our research programs required the preparation of hypophysectomized and orchidectomized rhesus monkeys. This afforded us the possibility to characterize and compare levels of the gonadotropin and inhibin subunit mRNAs in pituitaries from intact and castrate monkeys. Eighteen adult male monkeys, four of which had been bilaterally orchidectomized 5-9 months previously, were used in this study. Plasma concentrations of LH and FSH were, respectively, 188.5 +/- 5.3 and 246.8 +/- 25.2 ng/ml in the castrate monkeys and 25.8 +/- 4.5 and 4.1 +/- 1.1 ng/ml (mean +/- SEM) in the intact animals. Total pituitary RNA was hybridized to cDNA probes for cynomolgus monkey gonadotropin subunits (FSH beta, LH beta, and the common alpha-subunit) and for human inhibin subunits (alpha, beta B, and beta A) by Northern blot analysis, and mRNA levels were normalized by subsequent hybridization to cyclophilin. Each of the gonadotropin subunit probes hybridized to a single RNA species with the approximate sizes of 1.6 kilobases (kb; FSH beta), 0.7 kb (LH beta), and 0.8 kb (alpha). Levels of LH beta and alpha-subunit mRNAs in pituitaries from castrate monkeys were about 5- and 2-fold higher, respectively, than those in pituitaries from intact monkeys. FSH beta mRNA, on the other hand, was elevated about 27-fold in castrate monkeys [mean +/- SEM, 3176 +/- 408 cpm bound (n = 4 castrate) and 116 +/- 30 cpm bound (n = 8 intact]). Inhibin beta B-subunit mRNA was present in the monkey pituitary as a doublet of about 5 kb, and it was approximately twice as abundant in intact pituitaries as in castrate pituitaries. Hybridizations involving inhibin beta A cDNA revealed a faint band in the region expected for monkey beta A mRNA (6.5 kb) in three of six RNA samples from intact monkeys and a 0.3- to 0.4-kb mRNA species. mRNA encoding the inhibin alpha-subunit was undetectable by Northern blot hybridization. These results indicate that the postpubertal testis imposes an inhibition on the expression of the genes encoding FSH beta, LH beta, and glycoprotein hormone alpha-subunit and that this suppression of the FSH beta gene in the monkey is much greater than that in the rat. In addition, the monkey pituitary may be a source of activin, which may act locally to modulate FSH gene expression and secretion.

Stimulation of spermatogenesis in stalk-sectioned rhesus monkeys by testosterone alone.

Testosterone alone stimulated spermatogenesis in four adult, pituitary stalk-sectioned rhesus monkeys. Ten to 14 weeks after transection of the pituitary stalk, testicular volumes declined to about one fifth of the presurgical values. Serum LH levels declined precipitously, being undetectable 1 week postsurgery, and serum testosterone levels were indistinguishable from those of castrated male monkeys by the 5th week postsurgery. After a transient decline, serum PRL levels increased to high values in all four monkeys throughout the rest of the postsurgery period. Twelve weekly injections of 250 mg testosterone enanthate resulted in peak testosterone levels around 25-fold higher than presurgical levels. Estradiol levels increased about 4-fold over presurgical levels, and PRL also increased further during the treatment phase. Small ejaculates were produced by electrostimulation by the 5th week of treatment. Thereafter, the ejaculate weight increased. Sperm were found from the 10th week in all four monkeys. By the 13th week of treatment, sperm counts in three monkeys ranged from 17-60 X 10(6) sperm/ejaculate. The sperm counts continued to increase for the first 4 weeks after the cessation of the testosterone enanthate injections. Thereafter, the sperm counts declined, and all four animals produced azoospermic ejaculates between 10 and 31 weeks posttreatment. Moreover, testosterone levels declined slowly and nonuniformly among the four animals. Testicular volumes declined and were at the lowest levels 14 weeks posttreatment. Estradiol and PRL levels also declined posttreatment. It is concluded that testosterone alone can stimulate spermatogenesis in stalk-sectioned rhesus monkeys even in the face of high serum PRL and estradiol levels.

Reversible induction of azoospermia in rhesus monkeys by constant infusion of a gonadotropin-releasing hormone agonist using osmotic minipumps.

This study documents the long term, reversible, antireproductive effects of GnRH agonist treatment in adult male rhesus monkeys. Constant sc infusion of the GnRH agonist buserelin by osmotic minipumps over 20 weeks resulted in an initial rise in serum LH, followed by a decline to undetectable levels, indicating pituitary desensitization. This rise and fall of plasma LH was paralleled by serum testosterone concentrations. The RHLH response to an iv bolus injection of GnRH was completely abolished, and the corresponding Leydig cell response was also lost. Testicular volumes decreased markedly, and spermatogenesis was inhibited to azoospermic levels. Spontaneous and electrostimulated ejaculatory activities were lost under prolonged buserelin infusion. The animals were then supplemented with exogenous testosterone to reestablish ejaculatory behavior which had been curtailed under low circulating androgen levels, and azoospermia persisted. Such pronounced effects could not be demonstrated in our previous studies using daily or twice daily injections with higher agonist doses. When treatment was stopped after 20 weeks, the inhibitory effects of the GnRH agonist were reversible, and full pituitary and gonadal functions were recovered. It is concluded that constant infusion of GnRH agonist is far more effective in male rhesus monkeys than daily injections and that the suppressive effects of GnRH agonist application are fully reversible. Thus, a suitable primate model for further research on a GnRH agonist-based male contraceptive has been established.

Alkylating angiotensin II analogues: synthesis, analysis, and biological activity of angiotensin II analogues containing the nitrogen mustard melphalan in position 8.

Melphalan derivatives suitable for peptide synthesis, i.e., Boc-Mel and Mel-OBzl-HCl, have been prepared, and the integrity of their nitrogen mustard alkylating groups was examined by NMR, Volhard chlorine analysis, and colorimetric assay with 4-(p-nitrobenzyl)pyridine. By using the sensitive colorimetric assay, the stability of melphalan toward conditions commonly used for peptide synthesis, purification, and bioassay was evaluated. Further qualitative and quantitative assessment of the integrity of nitrogen mustard groups in angiotensin II was attempted in order to evaluate the significance of the observed biological results. [Ac-Asn1,Mel8]angiotensin II was a potent competitive antagonist of angiotensin II in vitro (rat uterus) but a transient and reversible inhibitor in vivo.

Role of the C-terminal carboxylate in angiotensin II activity: alcohol, ketone, and ester analogues of angiotensin II.

[Ac-Asn1, Val5]angiotensin II analogues containing a C-terminal alcohol (Phe-ol), methyl ketone (Pmk), methyl ester (Phe-OMe), or alpha-methyl methyl ester (Phe(alpha Me)-OMe) were prepared in order to examine the relative importance of COOH-mediated ionic vs. hydrogen bonding interactions in angiotensin activities. Based on the observation that only [Ac-Asn1,Phe-OMe8]AII (AII, angiotensin II) had significant activities (20% oxytocic and 13% pressor) in the rat, with all other analogues having negligible agonistic and antagonistic effects, it is concluded that ionic interaction of the C-terminal carboxylate with the receptor is necessary for angiotensin binding and that hydrogen bonding has little effect. Thus, the different potencies observed for the AII methyl ester and for various C-terminal analogues previously reported may simply reflect their relative abilities to generate the active carboxylate species in situ.

Three-dimensional quantitative structure-activity relationship of angiotesin-converting enzyme and thermolysin inhibitors. II. A comparison of CoMFA models incorporating molecular orbital fields and desolvation free energies based on active-analog and complementary-receptor-field alignment rules.

The utility of comparative molecular field analysis (CoMFA), a three-dimensional Quantitative Structure-Activity Relationship (3-D QSAR) paradigm, as a tool to aid in the development of predictive models has been previously addressed (Depriest, S.D. et al., J. Am. Chem. Soc. 1993, in press). Although predictive correlations were obtained for angiotensin-converting and thermolysin inhibitors, certain inadequacies of the CoMFA technique were noted. Primarily, CoMFA steric and electrostatic fields alone do not fully characterize the zinc-ligand interaction. Previously, this was partially rectified by the inclusion of indicator variables into the QSAR table to designate the class of zinc-binding ligand. Recent advances in molecular modeling technology have allowed us to further address this limitation of the preceding study. Using molecular orbital fields derived from semiempirical calculations as additional descriptors in the QSAR table, predictive correlations were produced based on CoMFA and molecular orbital fields alone--indicator variables no longer being necessary. Arbitrary information concerning the alignment of molecules under study within the active-site introduces ambiguities into the CoMFA study. Crystallographic information detailing the binding mode of several thermolysin enzyme inhibitors has previously been used as a guide for the alignment of additional, noncrystallized, inhibitors. However, this process was complicated by the lack of parameters for zinc in the molecular mechanical force field. Therefore, zinc-ligand interactions were ignored during the standard minimization procedure. The use of field-fit minimization using complementary receptor fields as templates is presented as a possible solution to the problem. Predictive correlations were obtained from analyses based on this method of molecular alignment. The availability of crystallographic data for thermolysin enzyme-inhibitor complexes allowed for an alternate definition of the CoMFA region. Herein, promising results from analyses using actual receptor active-site atom probe atoms are presented.