ABSTRACT

Following on from our recent evidence that Sertoli cells may regulate testicular interstitial fluid (IF) volume, this study has assessed whether depletion of specific germ cell types in vivo is associated with changes in recovered IF volume. Germ cell depletion was induced by either a single oral administration of 650 mg methoxyacetic acid (MAA)/kg or exposure of the testes to local heating (43 °C for 30 min). Treatment with MAA induced depletion or loss of most pachytene and later spermatocytes at 1–3 days and, because of maturation depletion, this resulted in the specific depletion of later germ cell types at 7–35 days. Testicular IF volume was unchanged at 1–7 days after MAA treatment but was increased significantly (P < 0·01) at 14 days and was nearly doubled (P< 0·001) at 21 days, before returning to control levels at 28–42 days. Serum LH (and FSH) levels were generally higher in MAA-treated rats, especially at 21 and 28 days, but there was no obvious correlation between LH levels and IF volume changes. Similarly, there was no relationship between IF volume changes and testicular weight or IF levels of testosterone. The increase in IF volume at 14–21 days after MAA treatment coincided with specific depletion of the later elongate spermatids (steps 14–19) and, when these cells reappeared in the testis, IF volume normalized.

This possible causal association was studied further in rats exposed to local testicular heating which, within 3 days, caused major depletion of pachytene spermatocytes and early (step 1–8) spermatids. However, testicular IF volume in heat-exposed rats did not change until 14 days, a time at which depletion of the later (step 9–19) spermatids first became evident; IF volume remained increased whilst these germ cells were absent or depleted. The pattern of change in IF volume in heat-exposed rats was not related to LH (or FSH) levels, which were raised at most time-points after heat treatment, nor to testicular weight which was decreased considerably at 3 days and declined progressively thereafter.

These data thus provide evidence that specific depletion of the most mature germ cell types (the elongate spermatids) is associated with specific changes in testicular IF volume, presumably via modulation of the secretion of vasoactive factors by the Sertoli cells. These findings also reinforce the growing evidence for the mutual interdependence of all of the cell types in the testis.

Journal of Endocrinology (1991) 128, 359–367

ABSTRACT

There is increasing evidence implicating growth factors in the regulation of spermatogenesis and in-vitro studies have shown that epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) interact with the gonadotrophins in regulating testicular function. In the present study, the effect of FSH, testosterone and GH treatment on serum IGF-I and intratesticular EGF and IGF-I concentrations in adult male hypophysectomized rats treated with ethane dimethane sulphonate (EDS) to destroy Leydig cells has been investigated. Hypophysectomy alone or followed by EDS treatment was associated with a significant increase in intratesticular EGF concentrations compared with normal controls (P<0·05). Treatment of hypophysectomized animals with a combination of GH, FSH and testosterone resulted in a return of intratesticular EGF concentrations to normal control levels. In a second group of animals treated with a 5 cm silicone elastomer implant of testosterone and FSH, intratesticular EGF concentrations were also not significantly different from those of normal controls. Following hypophysectomy alone or hypophysectomy and EDS treatment, a significant increase in circulating IGF-I concentrations occurred, which was only reversed following the administration of GH. In addition, increases in testicular IGF-I concentrations were evident in all treated animals compared with controls, an effect which was only partially reversed in animals treated with a combination of FSH and testosterone (1·5 or 5 cm implant). Similar results were obtained with a combination of GH and FSH or GH and testosterone, although GH alone had no effect on testicular IGF-I concentrations. A combination of GH, FSH and testosterone restored testicular IGF-I to concentrations not significantly different from controls. It is suggested that GH, FSH and testosterone can play an important role in regulating circulating and testicular IGF-I and testicular EGF in the adult rat, but the regulation of growth factor production is a complex process and a number of other factors are probably involved in vivo.

Journal of Endocrinology (1991) 129, 109–117

ABSTRACT

GH plays a major role in pubertal growth, effects mainly mediated by stimulation of insulin-like growth factor-I (IGF-I) production by the liver. However, the role of GH in the regulation of pubertal onset, spermatogenesis and fertility is still under debate. GH and FSH have, in addition, been implicated in the regulation of IGF-I production by Sertoli cells in a number of studies, although conflicting results have been reported. The interpretation of studies using GH-deficient mutant mice has been complicated by the presence of additional defects in the hypothalamic-pituitary-gonadal axis of these animals. We have therefore used GH-deficient mutant male rats with no other documented hormonal deficiencies to study the effect of GH administration on somatic and testicular development, circulating and testicular IGF-I concentrations and testicular histology.

Body weights in GH-deficient rats substituted with GH were not significantly different from untreated or GH-treated normal rats and were significantly higher than body weights in untreated dwarf rats. Similarly, circulating IGF-I concentrations in GH-treated GH-deficient rats were not significantly different from those in untreated or GH-treated normal rats but were significantly higher than circulating IGF-I concentrations in untreated dwarf rats. No differences in testicular IGF-I concentrations were observed in any of the groups studied. Testicular weights remained low in both untreated and GH-treated GH-deficient animals compared with control animals but spermatogenesis was qualitatively and quantitatively normal in all groups at the end of the observation period.

We conclude that GH does not play a major role in the regulation of testicular IGF-I production at puberty although we cannot exclude the possibility that the low but detectable levels of GH in the blood of mutant rats is sufficient to augment testicular responsiveness to gonadotrophins and therefore result in normal gonadal development.

Journal of Endocrinology (1991) 131, 279–285

ABSTRACT

Synchronization of spermatogenesis would provide an ideal model for the investigation of stage-dependent changes in the secretion of paracrine factors. In vitamin A-deficient animals subsequently injected with vitamin A, over 80% of seminiferous tubules were synchronized within three to five stages of the seminiferous cycle. Following replenishment of vitamin A, spermatogenic stages IV–VI (35 days), VI–VIII (38 days), IX–XII (41 days), I–IV (45 days) and V–VII (48 days) were observed. Despite synchronization of spermatogenesis at all stages, spermatogenesis was markedly impaired when evaluated in a quantitative fashion. At all times evaluated, numbers of round spermatids were reduced compared with age-matched controls. Numbers of pachytene spermatocytes reached control values only after 45 days of vitamin A replenishment. Elongate spermatids were almost totally absent up to 41 days after vitamin A replenishment. Testicular and epididymal weights were also reduced, although testicular weights showed a significant recovery over the time-course of the study. Serum and pituitary concentrations of LH and FSH were raised at the commencement of the study, with serum gonadotrophins returning to control values 48 days after vitamin A replenishment. Both testicular and serum testosterone concentrations in treated animals tended to be higher than in the controls.

Although synchronization of spermatogenesis was achieved, testicular testosterone concentrations did not reflect the stage-dependent cyclical changes observed in earlier studies. Testicular concentrations of testosterone were raised throughout the period of observation with the exception of animals synchronized around stages II–IV of the spermatogenic cycle. No correlation between the most frequent stages and intratesticular testosterone was found (r = 0·06, P > 0·1). Previous observations that testosterone concentrations are selectively increased at stages VII–VIII of the spermatogenic cycle are not supported by the present study.

Journal of Endocrinology (1989) 123, 403–412

ABSTRACT

In order to clarify further the role of FSH in the maintenance of spermatogenesis, adult rats were treated with purified human FSH (2 × 5 IU/day per rat), testosterone (1·5 cm silicone elastomer implant) or a combination of both hormones for 2 weeks following hypophysectomy.

After hypophysectomy alone, no elongate spermatids were observed and the numbers of pachytene spermatocytes and round spermatids observed were reduced when compared with untreated controls. Testosterone supplementation alone qualitatively maintained the formation of elongate spermatids in most seminiferous tubules, whilst in FSH-treated rats increased numbers of round spermatids and pachytene spermatocytes were observed when compared with hypophysectomized animals. Formation of elongate spermatids, however, did not occur under FSH treatment alone. A combination of FSH and testosterone treatment maintained spermatogenesis in an almost quantitative fashion. Numbers of pachytene spermatocytes and round spermatids were maintained at about 80% of levels seen in intact control animals. Treatment with FSH or testosterone alone maintained testis weights at significantly higher levels than those seen in hypophysectomized controls (FSH, 0·79 ± 0·05 g; testosterone, 0·81 ± 0·07 g; hypophysectomized, 0·50 ± 0·04 g). Animals treated with FSH and testosterone showed testis weights 20% below control values (1·22 ± 0·05 vs 1·51 ± 0·06 g; P <0·05). No increases in intratesticular or intratubular androgen concentrations or in testosterone: dihydrotestosterone ratios were observed in any of the hormone-treated groups when compared with hypophysectomized controls. In all hypophysectomized animals testicular androgen concentrations were reduced to <5% of control values.

The results obtained in this study suggest that FSH is involved in the maintenance of spermatogenesis in the adult rat and that the effects of FSH are not mediated through changes in intratesticular androgens. Low levels of testosterone in combination with FSH can almost quantitatively maintain spermatogenesis in adult rats.

Journal of Endocrinology (1989) 121, 49–58

ABSTRACT

The pubertal development of a novel GH-deficient mutant, the dwarf rat, has been evaluated. The establishment of normal spermatogenic function within small testes suggests that GH plays no role in spermatogenic function during puberty and adult life. However, a reduction in testicular size may reflect a reduced Sertoli cell population, suggesting that GH may be of importance in prepubertal testicular development.

Furthermore, marked differences between the homozygous dwarf rat and homozygous GH-deficient mouse mutants (e.g. Snell, Ames, pygmy and little mutants) have been demonstrated. It would appear that the GH deficiency in the rat mutant is far more specific for GH than those hitherto described in the mouse. In contrast to Snell dwarf mice mutants, pituitary and serum concentrations of FSH and LH are normal throughout pubertal development in the dwarf rat. Both spermatogenic function and seminal vesicle function develop normally, whilst in Snell dwarf mice spermatogenic function develops late in life and seminal vesicles remain infantile. Serum and testicular concentrations of androgen are also normal in dwarf rats. Homozygous dwarf rats have been shown to be fertile in previous studies; however, our observations suggest that despite spermatogenesis being qualitatively and quantitatively normal when assessed histologically, reduced testicular size seen in dwarf rats would lead to a reduced daily sperm output in these animals.

The dwarf rat represents a mutant in which the consequences of the selective depletion of GH may be studied on various endocrine systems. The reproductive axis appears to be only partially affected, at an early age, by GH deficiency.

Journal of Endocrinology (1990) 126, 193–201

ABSTRACT

Evidence derived predominantly from a series of in-vitro studies indicates that insulin-like growth factor-I (IGF-I) plays an important role as a paracrine and autocrine regulator within the testis. We investigated the effects of FSH substitution on spermatogenesis and testicular IGF-I content in rats treated with gonadotrophin-releasing hormone (GnRH) antagonist (ANT) and injected with ethane dimethane sulphonate (EDS), a Leydig cell toxin. FSH treatment partially prevented the marked decrease in intratesticular IGF-I in rats treated with only GnRH antagonist but not in GnRH antagonist + EDS-treated animals (controls, 191·0 ± 4·5; ANT, 80·1 ± 5·6; ANT + EDS, 81·6 ± 3·4; ANT + EDS + FSH, 86·3 ± 1·4; ANT + FSH, 137·7 + 7·3 (s.e.m.) ng/testis). Correlation analysis of testicular IGF-I content with the number of pachytene spermatocytes and round spermatids per cross-section revealed r values of 0·77 and 0·74 respectively (P < 0·001). We then analysed the same parameters in GnRH antagonist-treated rats which, in addition, received daily injections of the antiandrogen flutamide, in order to investigate the potential role of testosterone, as opposed to other Leydig cell products, in the regulation of spermatogenesis and testicular IGF-I. FSH treatment prevented regression of spermatogenesis in rats treated with GnRH antagonist alone but not in GnRH antagonist-and flutamide-treated rats. Testicular IGF-I content was altered in all treatment groups with the most marked changes observed in animals receiving GnRH antagonist and flutamide (FL) with or without FSH (5 or 10 IU/rat twice a day; FSH5 and FSH10) (ANT + FL, 150·32 ± 7·38; ANT + FL + FSH5, 165·28 ± 5·92; ANT + FL + FSH10, 160·17 ± 11·73 vs 464·51 ± 36·04 ng/testis for controls; P < 0·05). Those animals treated with GnRH antagonist and/or FSH had testicular IGF-I levels which were significantly lower than controls and higher than the three groups receiving flutamide and antagonist. A highly significant correlation was established between intratesticular IGF-I and the number of pachytene, round and elongated spermatids per cross-section (r = 0·8 for all three germ cell types). The data presented here provide direct in-vivo evidence for the importance of Leydig cell–Sertoli cell interactions in regulating testicular IGF-I content and spermatogenesis in the testis.

Journal of Endocrinology (1993) 137, 81–89