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OBJECTIVE Transdermal testosterone gel treatment is an effective androgen substitution therapy with several advantages over conventional substitution therapies. Whereas side-effects due to overdosing of hypogonadal patients are unlikely, testosterone gel application without protection may cause severe side-effects in other subjects (partners, family members) by contamination. Therefore, the risk of testosterone transfer of a newly developed 2.5% testosterone gel preparation was evaluated. DESIGN In two clinical randomized open single-centre studies on healthy male volunteers the percentage of testosterone remaining on the skin after gel application over time (n = 12) and the possibility of a transfer of testosterone to another person (n = 28) was evaluated. In the second study the endogenous testosterone production in the receiving subjects was suppressed by injecting 400 mg norethisterone enanthate (NETE). RESULTS After 8 h approximately 60% of testosterone applied to the skin could be recovered. When the skin had been previously washed with water, only about 14% of applied testosterone could be recovered. After intense skin contact with a volunteer who had applied testosterone before on his forearm, no increase in testosterone serum levels could be found in NETE-suppressed men. CONCLUSION Although considerable amounts of testosterone remain on the intact skin for several hours after evaporation of the alcohol vehicle, contamination of a second, especially female or prepubertal, subject causing side-effects seems very unlikely.

CD52 is a glycosylphosphatidylinositol-anchored glycoprotein secreted by the epididymis where it is incorporated into sperm membranes. The antigen is common to spermatozoa and lymphocytes, and its role is not known. Quantitative analysis using immunostaining with the monoclonal antibody CAMPATH-1G and flow cytometry indicated positive signals from approximately 80% of viable, washed ejaculated spermatozoa. Staining intensity increased after capacitation overnight, and decreased after short incubation with high density lipoprotein. After incubation of Percoll-washed spermatozoa with CAMPATH-1G, motility was reduced from 83 to 74% after 5 min and from 73 to 52% after 3.5 h. Swimming velocities were reduced by approximately 30% and linearity by 15% in 5 min, but no further decreases were observed over 3.5 h. After 20 min incubation with the antibody, cell viability was decreased by 10 and 20% in freshly washed and capacitated spermatozoa respectively. Comparison of fertile and infertile patients revealed no difference in the percentages of immunostained spermatozoa or in staining intensity and there were no differences in sperm labelling between normozoospermia and teratozoospermia. Compared with normozoospermia, percentages of stained spermatozoa were lower by 20 and 27% in asthenozoospermia and oligozoospermia respectively, whereas staining intensity in asthenozoospermia was less than half that in oligozoospermia. The correlation of percentages of stained spermatozoa with percentages of motile cells suggests the involvement of epididymal CD52 in the maturation of sperm function.

Bradykinin (BK) has been suggested to be an active substance in the disputed therapeutic use of kallikrein to improve semen quality. The effects of exogenous BK and its antagonist Hoe 140, which acts on one of the bradykinin receptors (BK2), were examined in two groups of patients attending the fertility clinic: those with asthenozoospermia (group I) and normozoospermia (group II). Bradykinin (10nM-1 microM) added to washed human spermatozoa had no effect on most kinematic parameters and caused only a marginal increase (7%) in curvilinear velocity at 50 nM in group I patients; however, this increase was not suppressed by concomitant addition of the BK antagonist. The bradykinin antagonist itself had no effect on the percentage motility or kinematic motility parameters of washed human spermatozoa in either group of patients. The motility of spermatozoa in semen was also unaffected by the presence of the bradykinin antagonist. It is concluded that bradykinin does not act exogenously on washed spermatozoa nor endogenously on spermatozoa in semen to stimulate motility via BK2 receptors, regardless of the initial quality of the sperm motility.

We reported previously that after a single injection of a gonadotrophin-releasing hormone (GnRH) antagonist to male monkeys, exogenous GnRH stimulated LH secretion in a time- and dose-dependent manner, indicating that GnRH antagonist-induced blockade of LH secretion resulted from pituitary GnRH receptor occupancy. The present study was performed to investigate whether GnRH can also restore a blockade of LH and testosterone secretion during chronic GnRH antagonist administration. Four adult male cynomolgus monkeys (Macacafascicularis) received daily s.c. injections of the GnRH antagonist [N-Ac-d-pCl-Phe1,2,d-TRP3,d-Arg6,d-Ala10]-GnRH (ORG 30276) at a dose of 1400–1600 μg/kg for 8 weeks. Before the GnRH antagonist was given and during weeks 3 and 8 of treatment, pituitary stimulation tests were performed with 0·5, 5, 50 and 500 μg synthetic GnRH, administered in increasing order at intervals of 24 h. At 8 weeks, a dose of 1000 μg GnRH was also given. All doses of GnRH significantly (P < 0·05) stimulated serum concentrations of bioactive LH (3- to 8-fold) and testosterone (2·6- to 3·8-fold) before the initiation of GnRH antagonist treatment. After 3 weeks of GnRH antagonist treatment, only 50 and 500 μg GnRH doses were able to increase LH and testosterone secretion. Release of LH was significantly (P < 0·05) more elevated with 500 μg compared with 50 μg GnRH. After 8 weeks, only the highest dose of 1000 μg elicited a significant (P < 0·05) rise in LH secretion. Basal hormone levels just before the bolus injection of GnRH were similar (P > 0·10–0·80). This finding eliminated the possibility that the increasing doses of GnRH had primed the pituitary thereby resulting in higher stimulatory effects of the larger doses of GnRH. In conclusion, the present data indicate that, even after prolonged exposure to a GnRH antagonist, the pituitary retains some degree of responsiveness to GnRH. This observation supports the view that the inhibitory effects of chronic GnRH antagonist treatment are also mediated, at least in part, by occupancy of the pituitary GnRH receptor rather than by receptor down-regulation. Journal of Endocrinology (1992) 133, 439–445

Animal experiments and clinical studies on the interactions between growth hormone (GH) and the male hypothalamic-pituitary-gonadal axis have predominantly concentrated on GH and sex steroid interactions in the regulation of growth and development, or on the metabolic effects of GH. In contrast, little attention has been paid to the possible effects of GH on spermatogenesis, although the first report dealing with this topic was published almost 30 years ago. The interactions of GH and its main mediator, insulin-like growth factor I (IGF-I), with the hypothalamic-pituitary-gonadal axis, and their role in spermatogenesis have recently been investigated using in vitro systems and different animal models (mice and rats). Using Leydig and Sertoli cell cultures, complex interactions between GH/IGF-I and the gonadotropins affecting differentiated cell functions, e.g. steroidogenesis and cell division, have been demonstrated at the cellular level. In vivo studies using immature and mature hypophysectomized rats and GH-deficient mutant male mice and rats indicate that IGF-I can play an important role in the regulation of steroidogenesis and spermatogenesis. Furthermore, although follicle-stimulating hormone and luteinizing hormone are the major regulators of testicular IGF-I production, GH may play an indirect role by potentiating the actions of the gonadotropins in regulating testicular IGF-I content. A large proportion of men presenting at male-infertility clinics are diagnosed as having idiopathic infertility. Further studies are necessary to investigate whether defects associated with GH and/or IGF-I effects in the testis are the cause of male infertility in a small group of these patients.

Epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) have been shown to regulate Leydig cell steroidogenesis in several species. We have investigated the effects, if any, of EGF and IGF-I on in vitro testosterone production of human Leydig cells. Interstitial cells or Percoll purified Leydig cells were isolated from the testes obtained from patients (n = 9) undergoing orchidectomies for treatment of prostate cancer and were cultured for different time periods with hCG, dibutyryl cAMP, EGF and IGF-I. Testosterone in the culture media was measured by radioimmunoassay. While EGF had a stimulatory effect on basal testosterone production of isolated interstitial cells or purified Leydig cells, IGF-I was ineffective. When the interstitial cells were cultured in the presence of hCG or EGF for 3, 6 or 24 h, the stimulatory effects of EGF on testosterone production were only evident after 24 h. On the other hand, hCG stimulated testosterone production at all time points (i.e after 3, 6, 24 h of incubation). When added in the presence of maximal concentrations of hCG and dibutyryl cAMP, EGF did not further enhance steroidogenesis. On the other hand, IGF-I potentiated the effects of hCG on testosterone production. These studies suggest that EGF and IGF-I may play a regulatory role in steroidogenic function of the human testes.

Morphometric study revealed that, at 40 days after the start of vitamin A replacement, A1 spermatogonia and preleptotene spermatocytes appeared in more than 70% of the whole mounts of seminiferous tubules of vitamin A-deficient rats. By 42 days, the appearance of these cell types was reduced by 50%, and A2 and A3 spermatogonia were predominant. By 46 days, A1-A3 spermatogonia appeared in less than 30% of the tubular length while A4, intermediate and B spermatogonia became the major cell types in the basement compartment of seminiferous tubules. The predominance of spermatogonia noted at given times was corroborated by higher frequencies of tubular cross-sections of stages in which that particular type of spermatogonium resides. These results indicate that seminiferous tubules of vitamin A-replaced-vitamin A-deficient rats are 'enriched' for particular stages. Tracing the development of [3H]thymidine-labelled preleptotene spermatocytes revealed normal kinetics of germ cell differentiation in these animals. Furthermore, the spermatogonial proliferations in the vitamin A-replaced-vitamin A-deficient rats were quantitatively normal. We suggest that vitamin A replacement may result in temporal suppression of the differentiation of A2-B spermatogonia, leading to a stimulation or synchronization of certain groups of undifferentiating spermatogonia which undergo active proliferation simultaneously. These synchronized populations of spermatogonia continue to proliferate and differentiate, thus resulting in the stage-enrichments noted at later times.

Objective: Testosterone-containing gels have improved testosterone substitution therapy, but they are associated with the risk of interpersonal transfer. Therefore, we tested a new hydroalcoholic 2.5% testosterone gel (TGW), which was removed by washing 10 min after administration. Design: The gel was applied to scrotal or non-scrotal skin in comparison to two 2.5 mg Androderm® patches in a randomised, three-arm, parallel-group, controlled multicentre trial over a period of 24 weeks. We included symptomatic hypogonadal men whose morning testosterone levels were n = 54) or 5 g to non-scrotal skin (n = 56) once daily; the patch group (n = 52) applied two patches/day. Dose titration was allowed. Results: Whereas serum testosterone levels and the pre-post changes of the areas under the curve of testosterone and free testosterone between weeks 0 and 24 indicated equivalent treatment success for the patch and scrotal groups, the dermal gel group was significantly superior to the other two groups. Questionnaires on sexual function, mood and quality of life did not differ significantly between study groups, nor were prostate volume, prostate-specific antigen (PSA) levels and prostate symptoms different. However, tolerability was much better in the gel groups than the patch group. Conclusion: Efficacy, safety and tolerability suggest TGW as a favourable treatment for hypogonadal patients.

The changes of reproductive functions, fertility and genetic risks of ageing females have been investigated relatively intensively. The state of knowledge about endocrine and reproductive changes in ageing men as well as possible age-related paternal genetic risks lags far behind. In general serum testosterone levels decrease with increasing age. In ageing men testis morphology and semen production can be absolutely normal. However, in some testes degenerative forms of germ cells can be observed. Fertility may be maintained up to a very high age. The genetic quality of sperm produced by older men may be reduced for several reasons, among which age-related increases in germ cell mutations, impairment of DNA-repair mechanisms and apoptotic processes are the most likely. The incidence of several autosomal dominant diseases, such as achondroplasia, polyposis coli, Marfan syndrome, Apert syndrome or basal cell naevi is associated with advanced paternal age, whereas there is no clear evidence for a paternal effect on structural or numeric chromosome anomalies. Hence even if offspring of older men have a slightly increased incidence of certain genetic diseases, the individual risk of such a new disease must be considered as extremely small, as the incidence of these diseases is very low.

14.3 Pharmacology of testosterone preparations 14.3.1 Oral administration 14.3.1.1 Unmodified testosterone 14.3.1.2 17 -methyltestosterone 14.3.1.3 Fluoxymesterone 14.3.1.4 Mesterolone 14.3.1.5 Testosterone undecanoate 14.3.2 Sublingual application 14.3.3 Buccal application 14.3.4 Nasal application 14.3.5 Rectal application 14.3.6 Intramuscular application 14.3.6.1 Testosterone propionate 14.3.6.2 Testosterone enanthate 14.3.6.3 Testosterone cypionate and testosterone cyclohexanecarboxylate 14.3.6.4 Testosterone ester combinations 14.3.6.5 Testosterone buciclate 14.3.6.6 Testosterone undecanoate 14.3.6.7 Testosterone decanoate 14.3.6.8 Testosterone microspheres 14.3.7 Subdermal application 14.3.7.1 Testosterone pellets 14.3.7.2 Testosterone microcapsules 14.3.8 Transdermal application

OBJECTIVE: The effects on male reproductive parameters after 1 year of treatment with recombinant human GH to the cynomolgus monkey were investigated. DESIGN: Twenty-four male cynomolgus monkeys were given daily subcutaneous doses of 0 (vehicle) (n=7), 0.4 (n=5), 2.0 (n=5) and 10.0 (n=7) IU/kg bodyweight for 52 weeks. At completion of the treatment period two control and two high-dose animals were left for a 12-week treatment-free period. METHODS: Before and during the treatment period and during the recovery period, sperm analyses, testicular volume measurements and hormone analyses of prolactin (PRL), LH, FSH, testosterone and IGF-I in serum, and analysis of serum antibodies against human GH were performed. Testicular morphology was monitored by biopsies, predose and on day 15 of the study, and with light microscopy on organ samples collected at time of death, at the end of the treatment, and during recovery periods respectively. RESULTS: Of all studied parameters, alterations were observed only in serum levels of IGF-I and PRL. IGF-I showed a dose-dependent increase throughout the treatment, with a normalisation during the treatment-free period. PRL decreased significantly in animals given 10.0IU/kg per day from week 14 of treatment and throughout the study but with a normalisation upon cessation of treatment. Spermatogenesis, as judged from semen analysis, testicular volume measurements and testicular morphology was not affected. CONCLUSION: This controlled preclinical study demonstrates that high doses of human GH do not alter male reproductive parameters in a non-human primate model.

The present chapter reviews current knowledge concerning hormonal regulation of gametogenesis in the primate testis. LH/testosterone and FSH are the prime regulators of primate spermatogenesis. Although either hormone is capable of stimulating all phases of the spermatogenic process including the formation of sperm, the combination of both hormones is necessary in most instances to achieve quantitatively normal germ cell numbers. Sertoli cell proliferation can also be induced by either hormone in juvenile monkeys. Evidence for differential effects of testosterone and FSH on gametogenesis, however, is lacking and a synergistic effect is observed when they are combined. Receptors for androgens and FSH occur exclusively on testicular somatic cells and, hence, the trophic effects of these hormones on germ cell numbers are indirect ones. Interestingly, both hormones seem to have a common target, the spermatogonial population but it is unknown how such an indirect albeit highly specific effect is mediated. Whether the trophic hormone action influences germ cell numbers via increased proliferation or decreased cell death or both remains to be seen. There is evidence to suggest that the local androgen requirements for primate spermatogenesis might be comparatively high.

Summary. The most widely used tests to detect seminal and serum sperm antibodies are the mixed antiglobulin reaction (MAR as recommended by WHO) and the tray agglutination test (TAT). It has been suggested that the prognostic significance of sperm antibody tests might be influenced by a concomitant reduction of sperm numbers and/or sperm motility. Furthermore, the relative sensitivity of these sperm antibody tests to detect sperm antibodies is not known. We therefore compared TAT, performed with serum and MAR results retrospectively for 565 infertile patients and MAR IgA and MAR IgG results for 1189 infertile patients. The association of TAT and MAR results with changes in sperm number, morphology and motility was assessed for 565 and 1185 patients, respectively. The influence of MAR and TAT results on sperm cervical mucus penetration test (SCMPT) results was investigated for 349 and 434 patients, respectively. Whereas only 23% of all MAR IgG positive patients were also MAR IgA positive, 82% of all MAR IgA positive patients were also MAR IgG positive. There was a significant (P < 0.0001) correlation between serum TAT, and MAR results. Positive MAR and TAT results were not associated with reductions in sperm number, motility and morphology. There was a significant correlation between MAR IgG and MAR IgA results and the sperm cervical mucus penetration test (SCMPT) results. According to these results, the MAR IgG would be sufficient as an initial screening for seminal sperm antibodies. MAR IgG negative patients with strong indication for immunologic infertility should also be investigated with the MAR IgA and the serum TAT. Serum and seminal sperm antibodies do not seem to influence male fertility via reductions of sperm number, motility and morphology. However because of their significant influence on SCMPT results, MAR IgG and MAR IgA results seem to be of prognostic significance.

In this study the localization and regulation of steady-state follistatin messenger ribonucleic acid (mRNA) levels in testicular cell cultures were examined with a solution-hybridization assay using a specific 32P-labelled cytosolic RNA antisense probe for follistatin and a 35S-labelled cytosolic RNA antisense probe for cyclophilin as internal standard. Testes from immature rats were dispersed with collagenase and fractionated in Sertoli and Leydig cell-enriched cultures. Follistatin mRNA was mainly localized to the Sertoli cell-enriched fraction and the expression of follistatin mRNA could be stimulated in vitro with fetal calf serum, epidermal growth factor or phorbol-12-myristate-1 3-acetate (an activator of protein kinase C), whereas follicle-stimulating hormone and forskolin (an activator of protein kinase A) had no effect. Neither prostaglandin E2, the synthetic glucocorticoid RU 28362 or all-trans-retinoic acid, which all regulate follistatin mRNA levels in non-testicular cell types, nor extracellur adenosine triphosphate (a purinergic receptor agonist) or testosterone had any obvious influence on follistatin mRNA levels in Sertoli cell-enriched cuitures. From this study it is concluded that Sertoli cells are likely to be the source of follistatin expression in the rat testis, that follistatin mRNA levels in Sertoli cell-enriched cultures are subjected to regulation by epidermal growth factor and the protein kinase C-dependent pathway but are not regulated by extracellular adenosine triphosphate, follicle-stimulating hormone, all-trans-retinoic acid, prostaglandin E2, forskolin, testosterone or the glucocorticoid RU 28362 and that the regulation of follistatin mRNA is sex- and tissue-specific.

In this paper the properties of a novel group of compounds, gonadotrophin-releasing hormone antagonists, are used to study the regulation of testicular function. These studies provide the basis for new developments in male contraception.

Scrotal temperature was monitored using a portable data recorder for periods of 24 h in six normal volunteers and 48 infertile patients with unilateral varicocele while subjects pursued their regular daily activities. Temperatures during sleep (TS) were generally higher than daytime values (TD), probably as a consequence of thermal insulation in bed. These diurnal variations were found to be less pronounced in the infertile patients than in volunteers (TS - TD = 0.29 degrees C +/- 0.06 degrees C vs 0.88 degrees C +/- 0.12 degrees C; P0.01). Moreover, scrotal temperatures at night of patients and volunteers were indistinguishable statistically, but were different during daytime hours. After successful ligation or embolization of the spermatic-vein in 16 patients, no change in scrotal temperature was observed. Although sperm counts were higher after treatment, this difference was not significant (67.4 +/- 17.2 x 10(6) vs 105.8 +/- 25.5 x 10(6); P0.05). The data support the view that varicocele-related damage to the testis results from a lack of adequate cooling, and that treatment does not normalize the temperature pattern.

Luteinizing hormone-releasing hormone (LH-RH) is produced in hypothalamic nuclei, travels via the portal blood to the anterior pituitary, and acts on gonadotropic cells by inducing the synthesis and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The gonadotropic hormones, in turn, regulate gonadal steroid production and gametogenesis. Reproductive functions, therefore, are critically dependent on LH-RH whose stimulatory effects are determined by its pulsatile release pattern (Knobil 1980) and by the availability of high-affinity binding sites at the pituitary site (Clayton and Catt 1981). Consequently, interference with the binding of native LH-RH to its pituitary receptors will provoke alterations of gonadal functions.

In replacing an endogenous hormone a safe general principle appears to be to mimic, as closely as possible, the normal concentrations of that hormone or its active metabolites (Cantrill et al. 1984). Following this principle testosterone treatment of male hypogonadism should avoid unphysiologically high testosterone serum concentrations to prevent possible side-effects or low concentrations to prevent androgen deficiency.

In order to evaluate the endocrine testicular and adrenal function of rhesus monkeys in vivo, and to compare this with the human situation, the effect of dexamethasone (Dxm), HCG and combined Dxm/HCG administration on the steroid levels was investigated in 8 adult male rhesus monkeys in November/December (= season of high testicular activity). Serum levels of cortisol, testosterone (T), androstenedione (A), 17-hydroxyprogesterone (17-OHP), oestradiol (Oe2), dehydroepiandrosterone (DHA) and its sulphate (DHA-S) were measured radioimmunologically. Basal T and DHA serum levels were similar to those found in man, whilst A levels amounted to 1/10 and DHA-S to 1/30 of human values. Oe2 and 17-OHP levels were higher than in man. T and A levels were not suppressed by Dxm and very high degrees of stimulation were reached after HCG. The HCG-induced increment in T was smaller under Dxm/HCG than under HCG alone. 17-OHP levels were reduced to 25% of basal levels under Dxm, but the incremental response to Dxm/HCG was the same as that without Dxm. DHA and DHA-S were 60 and 75% lower under Dxm, and the increment in DHA after Dxm/HCG was less than that after HCG. DHA-S did not respond to HCG. In conclusion, several important differences in basal steroid levels and in the response to dynamic testing were found in the rhesus monkey, as compared to man. The adrenal contributes 75% to circulating 17-OHP levels, and 60 and 75% respectively to DHA and DHA-S levels. A is produced solely by the testis in the rhesus monkey. As compared to man, the rhesus monkey testis in vivo synthesises T via both the Δ4 and Δ5 pathways. The Δ5 pathway appears to be dependent on adrenal steroid precursors.

The effect of a new analogue of the gonadotrophin-releasing hormone LH-RH, D-Ser(TBU)6-EA10-LH-RH, on the secretion of LH, FSH, as well as testosterone, oestrone, oestradiol, HGH

Summary Ligated tubules from the corpus epididymidis of men and monkeys were incubated in medium containing horseradish peroxidase (HRP) as a marker for fluid-phase endocytosis. HRP was localized by light and electron microscopy after 0, 15, 30 and 60 min of incubation. Movement between the cells was prevented by tight junctions, but bypass of this barrier was apparently achieved by an intracellular vesicular mechanism leading to a time-dependent appearance of HRP in the lumen. Uptake of HRP into basal cells and capture by the lysosomal apparatus of principal cells were also observed. HRP-filled vesicles also appeared in the basal, mid and apical cytoplasm of epithelial cells in the caput 1 h after injection of the tracer into the epididymal circulation of the monkey, suggesting that this pathway also operates in vivo.

The biological and immunological effects of immunisation against testosterone are reviewed and considered in regard to potential applications in male fertility control. In experimental animals active immunization with testosterone causes atrophy of the accessory reproductive glands, loss of sexual activity, increase in gonadotrophins, decrease in metabolic clearance rate of testosterone and prevention of fetal sexual differentiation. These findings can be described as “immunological castration”. Paradoxically, hyperplasia of the Leydig cells with excessively high testosterone levels in circulating blood occurs, while spermatogenesis remains intact. Thus infertility is achieved but at the expense of peripheral actions of testosterone. In addition, in actively immunized animals a high degree of immune complex nephritis is observed. Therefore, although a valuable research tool, immunization with testosterone does not represent a feasible approach to male fertility control.

The influence of sex, age, and liver function on the androgenmetabolizing capacity of the liver was investigated. To that end, testosterone was administered orally to healthy men, women and prepubertal boys as well as to men with liver dysfunction. While normal men (n = 17) and men with fatty liver (n = 9) showed no rise in circulating plasma testosterone levels and only a slight increase in androstenedione, a steep increase in both steroids was observed in men with liver cirrhosis (n = 7). Unlike adult men, boys (n = 7) and women (n = 6) also showed elevated testosterone levels after oral administration of this steroid. It is concluded that the hepatic capacity to metabolize testosterone is influenced by sex, stage of development, and liver function. It seems possible that peripheral concentration of testosterone after its oral administration can serve as a clinical test for liver function.

Pituitary–testicular relationships in mature male rats were investigated during the period of germinal involution after the induction of vitamin A deficiency (VAD). Vitamin A deficiency caused a decrease in testicular weight, a gradual increase in the incidence of delayed spermiation, increased phagocytosis of spermatids and pyknosis of germ cell nuclei in rats aged 80 to 110 days. Both basal and gonadotrophin releasing hormone (GnRH)-stimulated serum FSH concentrations were increased by 100 days of age. During the same period, the per cent increment in GnRH-stimulated FSH secretion, pituitary FSH concentration and LH secretion remained unchanged. These results suggest that the increased serum FSH may mark specifically an alteration in the germinal epithelium. By 140 days of age, spermatogenic activity in the rats with VAD was limited to the spermatogonial proliferations so that only Sertoli cells, spermatogonia and preleptotene spermatocytes remained. At this time hypersecretion of FSH persisted while the per cent increment of GnRH-stimulated FSH secretion decreased. Concomitantly, basal and GnRH-stimulated LH concentrations were also increased in the presence of normal serum testosterone. These results indicate that a complete cessation of spermatogenesis beyond preleptotene spermatocytes is associated with a change in the secretion of both FSH and LH. The relationship between serum LH and testosterone was normal until at least 110 days of age. By 140 days the ratio between basal LH and basal testosterone, and between total LH and total testosterone, after GnRH administration, increased in the rats with VAD. These changes may be caused by a hyporesponsiveness of the Leydig cells which may, in turn, be attributed to the cessation of spermatogenesis. J. Endocr. (1984) 100, 33–41

Before immunisation with hormones can be applied in therapeutic medicine, the possible immunological side effects should be investigated. Therefore the kidneys of 20 rabbits immunised with testosterone-BSA conjugates were examined 4 to 30 weeks after immunisation for the presence of immune complex precipitation and glomerulonephritis. Eighty-five per cent showed differing degrees of precipitation and 50 % had an exudative glomerulonephritis. None of the kidneys from normal control rabbits or those immunised with complete Freund's adjuvant or BSA showed any signs of immune complex precipitation, although high anti-BSA titres were present in the BSA-immunised animals (1/3000 – 1/25 000). BSA antibodies could not be detected in the rabbits immunised with testosterone-3-BSA. In the light of these findings active immunisation with steroid-BSA conjugates would not be advisable as a means of therapy.

Active immunization with steroids leads to neutralization of their biological effects since the hormone in serum is rendered unobtainable to receptors by binding to circulating antibodies. Therefore, if the steroid plays a role in gonadal or adrenal feedback control, immunization must result in increased gonadotrophin or ACTH secretion, inducing gonadal or adrenal hypertrophy and hyperfunction. We exploited this phenomenon to study the importance of various steroids in gonadal or adrenal feedback control. Groups of rabbits were immunized with different steroid conjugates and the morphological and functional alterations of the testes and adrenals were recorded. We thereby confirmed the role of testosterone and corticosterone in testicular and adrenal feedback control. Estradiol is also active in testicular feedback, but appears to act only on LH and not on FSH. In addition to corticosterone, dehydroepiandrosterone and aldosterone are of importance in adrenal feedback. Immunization against dihydrotestosterone and androstenedione yielded equivocal results because of high cross reaction of the respective antisera with other androgens. In general, the specificity of the antisera is a limiting factor of this immunological approach for the study of feedback control mechanisms.

Washed human spermatozoa from 21 individuals with an average motility of 60% (quality index 277 +/- 16) had an endogenous ATP generation of 7.5 +/- 3.4 nmole/10(8) spermatozoa. The ATP concentrations in spermatozoa from 16 patients with severely impaired motility of 26% (quality index 98 +/- 13) was 16.9 +/- 9.9 nmole/10(8) spermatozoa. There was no correlation between ATP content and motility in either group. Sperm penetration into blood serum type AB, Rh-positive, was evaluated using a capillary tube penetration test. The penetration was graded with a maximum score of 14. Spermatozoa with an initial motility of 60% reached a score of 10 +/- 0.7. After addition of 20 mmole of ATP the score was significantly improved to 13 +/- 0.3. Compared with these results spermatozoa with an average motility of 26% reached a score of 4 +/- 1. Exogenous ATP (20 mmole) increased the score to 8 +/- 1.0. In both groups reduced glutathione had a negative effect. Human spermatozoa with high and low motility are capable to synthesize ATP. A dysfunction of the phosphorylating particles in the mitochondria appears not to be associated with low sperm motility.

Non-glucoregulatory hormones (T4, T3, rT3, TSH and testosterone) were studied by radioimmunoassay in juvenile-type diabetics in moderate control and in ketosis due to insulin withdrawal and in age matched "normals" during a mild prolonged exercise test. The basal serum hormone levels revealed the following findings: Serum testosterone was markedly lower in diabetics than in normals ( 177 +/- 24 resp. 618 +/- 52 ng/dl). This is in contrast to other studies, but it may reflect decreased testicular function due to an early, clinically not apparent atherosclerotic disease. Serum T3 was significantly lower in diabetics than in normals (110 +/- 16 resp. 145 +/- 19), suggesting an early "low T3-syndrome" in juvenile-type diabetics. However, increased serum rT3 levels were not observed, and serum T4 and TSH were normal. Mild prolonged exercise had no major effects on these nonglucoregulatory hormones. In juvenile-type diabetics the degree of metabolic control had no influence on the response of the mentioned hormones. However, an increased cortisol/testosterone ratio in ketotic diabetics in the basal state with a further increase during exercise was demonstrated, indicating an aggravation of the catabolic state in these patients during exercise.

Plasma testosterone and androstenedione levels in men were measured after oral administration of free testosterone and testosterone undecanoate. Both androgens were determined by simultaneous, specific radioimmunoassays after separation and isolation by thin layer chromatography. While free unesterified testosterone had no effect on plasma androgen levels, a striking increase of both testosterone and androstenedione levels was noted after administration of testosterone undecanoate, which is otherwise only achieved by parenteral testosterone application. This effect of testosterone undecanoate is probably due to absorption via the lymph rather than via the portal vessels so that peripheral circulation is reached before metabolism in the liver. Testosterone undecanoate promises to be an effective medication for oral androgen replacement.

Oestrone, oestradiol-17β and testosterone in serum were measured during a LH-RH infusion test and a Leydig cell function test with HCG in healthy men and in patients with primary or secondary hypogonadism. Following infusion of LH-RH increases of LH (+112%), oestradiol (+62 %) and testosterone (+51 %) were observed in normal men, while oestrone remained unchanged. Of the patients only those with Klinefelter's syndrome showed a significant increase in testosterone and oestradiol after LH-RH infusion. During the Leydig cell function test oestradiol and testosterone exhibited a largely parallel pattern under normal and pathological conditions as well as in subjects with experimental hypogonadism. Augmentations of plasma oestrone were usually smaller than those for oestradiol.

A new method for the investigation of hypothalamo-pituitary-gonadal and adrenal feedback control mechanisms based on the biological neutralization of gonadal and adrenal steroids by active immunization is proposed. The regulatory influence of a given steroid in the feedback control is proved when reduction of the free, biologically active fraction of this steroid caused by antibody binding induces a positive response of the pituitary, thus effecting gonadal or adrenal hypertrophy and hyperfunction. The advantages and limitations of the new model are demonstrated by the effects of active immunization of rabbits with cortisol (F), aldosterone (Aldo), dehydroepiandrosterone (DHA), androstenedione (Δ4-A), testosterone (T), 5α-dihydrotestosterone (5α-DHT), 5β-DHT and oestradiol (E2). In the immunized animals and in a control group serum concentrations of total corticosteroids (TC), DHA, T, Δ4-A, E1, E2, LH and FSH, the percentage of binding of steroids in serum and the specificity of the antisera are determined. The testes are evaluated by histometry and the nuclear volume of the adrenocortical and Leydig cells is measured.