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Background Many environmental chemicals have been proposed as endocrine disruptors in the fetal testis. Studies in rats have demonstrated reproductive abnormalities after in-utero exposure to environmental chemicals (eg, phthalates) or pharmaceutical drugs such as paracetamol. Whether such effects also occur in the human fetal testis has been difficult to determine. We have recently demonstrated that xenografting of human fetal testis tissue results in normal seminiferous cord formation and cellular development/function. We aimed to determine the effects of proposed endocrine disruptors (eg, phthalates, paracetamol) on the human fetal testis using a xenograft approach. Methods Human fetal testes (14–20 weeks' gestation, n=17) obtained from elective terminations were xenografted into nude mice. Host mice received di-n-butyl phthalate (500 mg/kg/day), paracetamol (350 mg/kg/day), or vehicle during the grafting period. Testosterone production was determined by measurement of host animal seminal vesicle weight. Morphological and immunohistochemical analysis with a range of markers was performed to investigate seminiferous cord structure, steroidogenesis, and cellular development. Findings Exposure to paracetamol resulted in a significant reduction in seminal vesicle weight (mean 13·6 mg [SD 8·5] vs 10·0 [5·4], p vs 23 [11·3], p Interpretation These results suggest that paracetamol may impair testosterone production in the human fetal testis, whereas phthalates do not. This highlights important differences between rat and man in terms of the effects of chemical exposure on the developing testis. Funding Wellcome Trust and British Society for Paediatric Endocrinology and Diabetes.

Testosterone is required for normal development of the male reproductive tract. Synthesis of testosterone occurs in the Leydig cells and is dependent upon the expression of several enzymes, including cytochrome P450 17alpha-hydroxylase/C17-20-lyase (P450c17), which is highly regulated within the testis. The aim of the present study was to investigate whether maternal exposure to estrogenic chemicals was able to affect Leydig cell function in the developing male fetus at the time of masculinization. Pregnant rats were injected sc with diethylstilbestrol (DES; 100 or 500 micrograms/kg), 4-octylphenol (OP; 100 or 600 mg/kg), or vehicle (oil, control) on days 11.5 and 15.5 postcoitum. Doses were chosen to reflect the reported estrogenic potency of the chemicals in vitro. On day 17.5, fetal testes were fixed before performing in situ hybridization and immunocytochemistry, used for extraction of RNA, or homogenized in phosphate buffer for determination of 17alpha-hydroxylase enzyme activity. There was no difference between fetuses from control and treated mothers in either the overall histology of the testes or the apparent number of Leydig cells, as determined by immunocytochemistry with an antibody directed against 3beta-hydroxysteroid dehydrogenase. However, there was a consistent and striking reduction in the amount of P450c17 detected by immunocytochemistry in testes from the groups given the higher dose of DES or OP. These observations were supported by measurement of 17alpha-hydroxylase activity, which was significantly reduced compared with that in controls (6.25 +/- 0.65 pmol/testis x min) in fetuses from animals treated with 100 micrograms/kg DES (4.27 +/- 0.39; P0.05), 500 micrograms/kg DES (1.4 +/- 0.47; P0.001), or 600 micrograms/kg OP (4.25 +/- 0.33; P0.05). RT-PCR and in situ hybridization revealed that these changes were mirrored by reductions in P450c17 messenger RNA in testes from fetuses from treated mothers compared with control levels. In conclusion, maternal treatment with either a potent sythetic estrogen (DES) or a putative environmental estrogen (OP) results in reduced expression of the messenger RNA and protein for P450c17 in fetal Leydig cells. These results, therefore, provide a mechanism by which inappropriate exposure of the fetus to estrogenic chemicals might have an adverse effect on fetal steroid synthesis and masculinization.

Androgens are essential in fetal life for development of the male phenotype (1) and in the adult for maintenance of normal spermatogenesis (2). Androgen action is mediated by a specific intracellular receptor expressed in target tissues that binds testosterone and dihydrotestosterone with high affinity (3). Ligand binding is essential for activation of the receptor, which then binds to specific targets on DNA resulting in modulation of the level of gene expression (4). Recently we have successfully used immunocytochemistry, following microwave antigen retrieval, to demonstrate that androgen receptor (AR) expression in adult rat Sertoli cells (SC) occurs predominantly at stages II-VII of the spermatogenic cycle, with highest levels at stage VII (5)—results in agreement with other investigators (6). In our study AR immunostaining was also detectable in peritubular myoid cells, arterioles, and Leydig cells, but not in germ cells (5).

As in every organ in the body, mechanisms for the delivery and distribution of nutrients, hormones, and other messengers operate within the testis. In contrast to other organs (excepting the brain), the testis has a much greater dependence on these transport systems because of its unusual anatomy. The fact that the bulk of the testis is composed of the avascular seminiferous tubules, which have a very high energy/nutritional demand because of the proliferating germ cells, means that extravascular transport systems have to be highly developed if normal testicular function is to be maintained. This is probably why the intertubular spaces of the testes of most species contain abundant interstitial fluid (IF) (1), as it is this fluid that must transport factors from the bloodstream to the seminiferous tubules, Leydig cells, etc. However, IF cannot deliver nutrients and other factors directly to most of the developing germ cells because these are sequestered behind the “closed doors” of the inter-Sertoli cell tight junctions. Therefore, another delivery system, seminiferous tubule fluid (STF), is brought into play and is responsible for the transport of most factors from the Sertoli cells to the germ cells as well as the transport of spermatozoa out of the testis. There is also a fourth transport highway, testicular lymph, although its precise functions in the testis are not very clear (2).

This study assessed whether exposure of male rats to two estrogenic, environmental chemicals, 4-octylphenol (OP) and butyl benzyl phthalate (BBP) during gestation or during the first 21 days of postnatal life, affected testicular size or spermatogenesis in adulthood (90-95 days of age). Chemicals were administered via the drinking water or concentrations of 10-1000 micrograms/l (OP) or 1000 micrograms/l (BBP), diethylstilbestrol (DES; 100 micrograms/l) and an octylphenol polyethoxylate (OPP; 1000 micrograms/l), which is a weak estrogen or nonestrogenic in vitro, were administered as presumptive positive and negative controls, respectively. Controls received the vehicle (ethanol) in tap water. In study 1, rats were treated from days 1-22 after births in studies 2 and 3, the mothers were treated for approximately 8-9 weeks, spanning a 2-week period before mating throughout gestation and 22 days after giving birth. With the exception of DES, treatment generally had no major adverse effect or body weight: in most instances, treated animals were heavier than controls at day 22 and at days 90-95. Exposure to OP, OPP, or BBP at a concentration of 1000 micrograms/1 resulted in a small (5-13%) but significant (p < 0.01 or p < 0.0001) reduction in mean testicular size in studies 2 and 3, an effect that was still evident when testicular weight was expressed relative to body, weight or kidney weight. The effect of OPP is attributed to its metabolism in vivo to OP. DES exposure caused similar reductions in testicular size but also caused reductions in body weight, kidney weight, and litter size. Ventral prostate weight was reduced significantly in DES-treated rats and to minor extent in OP-treated rats. Comparable but more minor effects of treatment with DES or OP on testicular size were observed in study 1. None of the treatments had any adverse effect on testicular morphology or on the cross-sectional area of the lumen or seminiferous epithelium at stages VII-VIII of the spermatogenic cycle, but DES, OP, and BBP caused reductions of 10-21% (p < 0.05 to p < 0.001) in daily sperm production. Humans are exposed to phthalates, such as BBP, and to alkylphenol polyethoxylates, such as OP, but to what extent is unknown. More detailed studies are warranted to assess the possible risk to the development of the human testis from exposure to these and other environmental estrogens. Images Figure 1. Figure 2. Figure 3. A Figure 3. B Figure 3. C Figure 3. D Figure 4.

In line TGR(mRen2)26 transgenic rats (TGR26) bearing a randomly inserted additional renin transgene, the males, but not the females, were found to be infertile. Tissue was obtained from TGR26 males and littermate controls after perfusion fixation, and the morphology of the testes and epididymides was examined. Testis size was normal as was gross morphology, but careful examination revealed that the release of many spermatozoa at stage IX of the spermatogenic cycle was impaired. In addition, the process of cytoplasmic elimination was abnormal, as cytoplasmic fragments of elongate spermatids were present in the epididymis. In TGR26 males, seminiferous tubule lumen size was significantly larger (p < 0.001) than in littermate controls, a difference that was most marked at stages IX-XIV--an effect that could be related to the retention of spermatozoa. In situ hybridization confirmed that expression of renin mRNA could be detected in testes of TGR26 rats but not in normal controls or in a fertile line (TGR27) of rats bearing the same transgene. Immunocytochemistry and in situ and Northern hybridization were used to elucidate the pattern of expression of genes that previous studies have implicated in the process of sperm maturation and/or release. Of the gene products examined (sulphated glycoproteins 1 and 2 [SGP-1, SGP-2], transition proteins 1 and 2 [TP-1, TP-2], urokinase, and cyclic protein 2 [CP-2]), none showed any major change in the pattern of expression compared with that in controls. We postulate that TGR26 transgenic male rats may be infertile because the expression of a gene (or genes) involved in the process of cytoplasmic elimination and/or sperm release has been disrupted by the presence of the transgene close to or within the gene(s). Future planned studies will involve determination of the insertion site(s) and ultrastructural analysis of the final phases of spermiogenesis.

The objective of this study was to identify the early (after 4 and 24 h) effects of short-term local testicular heating (43 degrees C for 30 min) on the secretion of proteins by seminiferous tubules isolated from adult rats at stages II-V, VI-VIII or IX-XII of the spermatogenic cycle, and cultured in vitro for 24 h with [35S]methionine. Incorporation of [35S]methionine into secreted and intracellular proteins was assessed and the pattern of protein secretion was evaluated using two-dimensional SDS-PAGE. Seminiferous tubules isolated from control rats exhibited the characteristic, androgen-dependent increase in protein secretion at stages VI-VIII. At 4 h after exposure to local testicular heating, seminiferous tubules at these stages showed a significant increase (P < 0.001) in the overall incorporation of [35S]methionine into secreted proteins, whereas seminiferous tubules at stages II-V and IX-XII showed no significant change. In marked contrast, seminiferous tubules isolated from rats 24 h after local testicular heating showed a significant decrease in the incorporation of [35S]methionine into secreted proteins at stages VI-VIII (P < 0.001) and to a lesser extent at IX-XII (P < 0.05), whereas seminiferous tubules at stages II-V showed no change in incorporation. Prior treatment to maintain normal intratesticular concentrations of testosterone in heat-exposed rats failed to prevent these changes. Similar results were obtained when incorporation of [35S]methionine into intracellular proteins was evaluated 4 and 24 h after exposure to local testicular heating.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens are essential for the maintenance of normal spermatogenesis in the rat. We assessed the sites, developmental pattern, and hormonal control of androgen receptors (AR) in the rat testis. Adult male rats were studied after 1) no treatment; 2) ethane dimethane sulfonate (EDS), which eradicates Leydig cells and endogenous testosterone (T); 3) EDS plus T replacement beginning at the time of EDS administration; or 4) methoxyacetic acid, which leads to the loss of specific germ cell types. Testes were also obtained from normal immature rats (aged 5, 14, 16, 21, 28, 31, 35, 38, and 45 days). After microwave antigen retrieval, immunohistochemistry was performed using a rabbit polyclonal antibody (Novocastra) raised against a peptide unique to the N-terminal region of the AR and detection with biotinylated swine antirabbit immunoglobulin G, avidin-biotin complex/alkaline phosphatase, and nitroblue tetrazolium salt (NBT)/5 bromo-4-chloro-3-indolylphosphate (BCIP) substrate. In adults, nuclear immunostaining of Sertoli cells (SC) increased progressively in intensity from stages II through VII of the spermatogenic cycle, and then declined precipitously during stage VIII to become barely detectable in stages IX-XIII. Prominent AR immunostaining was also evident in peritubular myoid cells, arterioles, and interstitial cells; staining in these cells did not vary with the stage of the cycle of the adjacent tubules. EDS caused a severe loss of AR immunostaining in all cell types. Replacement of T in EDS-treated animals resulted in a pattern of AR immunostaining comparable to that in controls, although staining intensity was reduced. Methoxyacetic acid administration did not affect the pattern of AR staining. In immature rats, peritubular myoid cell immunostaining was prominent from day 5; SC staining was detectable on day 5, increased in intensity with age, and became stage dependent between days 21-35. The following conclusions were reached. 1) Immunohistochemically detectable AR expression in SC occurs predominantly in stages II-VII of the spermatogenic cycle, with highest levels at stage VII. 2) AR immunostaining is also prominent in peritubular myoid cells, arterioles, and Leydig cells (but not in germ cells), but is unrelated to the stage of adjacent tubules. 3) Endogenous T and/or its metabolites control the expression of AR in the testis. 4) AR immunostaining is detectable by day 5 of age and becomes stage specific in SC between days 21-35.

Hormonal regulation of the expression of mRNA transcripts for cAMP response element-binding protein (CREB) and cAMP response element modulator (CREM) during spermatogenesis was studied in the adult rat testis. Northern analysis of CREB and CREM identified two mRNA transcripts for CREM (2.4 and 1.6 kb) and one transcript for CREB (2.0 kb). Analysis of mRNAs from isolated testicular cells by reverse transcriptase polymerase chain reaction (RT/PCR) showed that CREM mRNAs were expressed by the germ cells but not the Sertoli or interstitial cells, whereas CREB mRNA was located in germ cells, Sertoli cells, and interstitial cells. RNA was isolated and analyzed from the testes of 1) rats treated for 24 h with FSH, 2) rats in which androgen withdrawal had been induced by ethane dimethane sulphonate (EDS) treatment 6 days earlier (EDS-treated), 3) EDS-treated rats supplemented with testosterone (EDS + T), or 4) intratesticular administration or dibutyryl cAMP (dbcAMP) in the preceding 24 h. CREM mRNA transcript expression was found to be decreased after all of these treatments in samples from intact testis and from isolated cells. Expression of the CREB transcript was also decreased by EDS-induced androgen withdrawal, but not by FSH or EDS + T. In situ hybridization of paraffin-embedded testis sections probed with digoxigenin-labeled riboprobes confirmed the localization of CREB and CREM mRNA to the same cell types as found with RT/PCR. No stage-dependent expression of CREM mRNA transcripts could be observed. Hybridization of the CREB probe was highest around the base of stage VII-VIII tubules, and this was shown to be androgen-dependent. The data presented suggest that regulation of the expression of CRE-binding protein mRNAs in Sertoli and germ cells during spermatogenesis is dependent on both androgen and FSH. However, the effects of androgen or FSH on the regulation of CRE-binding protein mRNAs are different.

Summary This study has assessed the effect of age on protein secretion by seminiferous tubules (ST) isolated from rats and their response to Sertoli cell toxicants. ST were isolated from immature (aged 28 days), late pubertal (aged 45 days) and young adult (aged 70 days) rats and cultured in vitro for 24 h with 35S-methionine in the presence or absence of FSH (1 mg/ml), m-dinitrobenzene (m-DNB) or nitrobenzene (NB) (both at 10-4M). Incorporation of 35S-methionine into newly synthesized proteins in the culture medium (secreted proteins) was assessed and the pattern of protein secretion evaluated using two-dimensional sodium dodecylsulphate polyacrylamide gel electrophoresis (2-D SDS-PAGE). These data were compared with those obtained using cultures of immature rat Sertoli cells + germ cells (SC+GC). Addition of FSH, m-DNB or NB in vitro either had no effect (NB) or had a small stimulatory effect (FSH and m-DNB) on the incorporation of 35S-methionine into overall secreted proteins by ST isolated from immature rats. At the same doses, addition of either FSH, m-DNB or NB to SC+GC co-cultures resulted in increased incorporation of radiolabel into secreted proteins in all instances. In contrast, the same additions to ST isolated from adult rats resulted in a 20–34% decrease in the overall incorporation of 35S-methionine into secreted proteins. ST isolated from late pubertal rats showed a similar response to adult rats except that the decreases in incorporation induced by FSH, m-DNB and NB were smaller. Analysis by 2-D SDS-PAGE revealed considerable age-dependent differences in the proteins secreted by ST from immature and adult rats, of which 13 were identified as being of potential importance. Most of these proteins were prominent secretory products of ST from adult rats, but were minor or non-detectable products of cultures of ST or SC+GC from immature rats. Most of these proteins disappeared or decreased in abundance after culture of ST with m-DNB or NB. Two proteins showed the reverse pattern, being more prominent secretory products in immature than mature rats, and their secretion was unaffected or was increased by toxicant exposure. These results demonstrate that there are major age-dependent differences in the secretion of total and specific proteins by isolated ST and that these are probably related to changes in the germ cell complement with age. The susceptibility of many of these proteins to perturbation by m-DNB and NB may explain the adverse effects of these compounds on spermatogenesis. Finally, the present results suggest strongly that studies of the regulation of spermatogenesis should not use cells/tissue isolated from immature rats.

The incidence of disorders of development of the male reproductive tract has more than doubled in the past 30-50 years while sperm counts have declined by about half. Similar abnormalities occur in the sons of women exposed to diethylstilbestrol (DES) during pregnancy and can be induced in animals by brief exposure to exogenous oestrogen/DES during pregnancy. We argue that the increasing incidence of reproductive abnormalities in the human male may be related to increased oestrogen exposure in utero, and identify mechanisms by which this exposure could occur.

Becoming a male is ultimately determined by androgen-induced masculinisation. Disorders of this, resulting in hypospadias (abnormal penis development) or cryptorchidism (undescended testes), are common disorders in humans. Together with adult onset disorders (low sperm counts, testis germ cell cancer) hypospadias and cryptorchidism may constitute a testicular dysgenesis syndrome (TDS) in humans, with a proposed common fetal origin that may involve deficiencies in androgen production or action. As animal evidence has shown that anti-androgenic endocrine disruptors (EDs) can induce TDS-like effects, concern has grown regarding their potential involvement in human TDS disorders. We have developed a rat model of TDS, in which we have begun to dissect the fetal mechanisms that underlie TDS disorders. Using another rat model in which androgen action is blocked by the anti-androgen flutamide, we have recently identified a ‘male programming window’, which available evidence suggests occurs also in the human fetus (between ∼8 and 12 week's gestation). Androgens must act within this programming window to set up later correct development of the reproductive tract and genitalia. Surprisingly, androgen-driven (abnormal) masculinisation of females is confined to the same programming window as for normal males. Blocking androgen action only within the programming window induces hypospadias and cryptorchidism and alters penile length in males; these all correlate with reduction in anogenital distance (AGD), which provides a non-invasive, lifelong read-out of androgen exposure in the programming window (but not later in gestation). As AGD is measurable in human newborns/neonates, it may predict adult onset TDS disorders and provide clinically important insights into reproductive tract masculinisation and its disorders. With this new understanding we have also begun to explore the role and timescale of effects of androgens in regulation of penile development and size. We show in the rat, that AGD predicts fetal and adult testis size (and thus sperm production), but the mechanism underlying this is unknown as it is not explained by effects on Sertoli cell number, as androgen regulation of Sertoli cell proliferation extends beyond the ‘male programming window’ (when AGD is determined); this relationship is of potential importance in the context of fetal origins of low sperm counts in humans. These findings strongly support the view that deficient fetal androgen action is a key feature of TDS. We postulate that delayed onset of fetal testosterone production may also be important in TDS. These new findings have considerable implications with regard to the time-window of susceptibility of the fetal male to disruption by EDs.

Androgen-mediated wolffian duct (WD) development is programmed between embryonic d 15.5 (e15.5) and 17.5 in male rats, and WD differentiation has been shown to be more susceptible to reduced androgen action than is its initial stabilization. We investigated regulation of these events by comparing fetal WD development at e15.5–postnatal d0 in male and female androgen receptor knockout mice, and in rats treated from e14.5 with flutamide (100 mg/kg/d) plus di-n(butyl) phthalate (500 mg/kg/d) to block both androgen action and production, testosterone propionate (20 mg/kg/d) to masculinize females, or vehicle control. In normal females, WD regression occurred by e15.5 in mice and e18.5 in rats, associated with a lack of epithelial cell proliferation and increased apoptosis, disintegration of the basement membrane, and reduced epithelial cell height. Exposure to testosterone masculinized female rats including stabilization and partial differentiation of WDs. Genetic or chemical ablation of androgen action in males prevented masculinization and induced WD regression via similar processes to those in normal females, except this occurred 2–3 d later than in females. These findings provide the first evidence that androgens may not be the only factor involved in determining WD fate. Other factors may promote survival of the WD in males or actively promote WD regression in females, suggesting sexually dimorphic differences in the preprogrammed setup of the WD.

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

This study has assessed whether depletion of specific germ cell types is able to alter the secretion of immunoactive inhibin by adult Sertoli cells in vivo and in vitro. Pachytene and later spermatocytes were depleted (80-100%) by a single administration of methoxy acetic acid (MAA; 650 mg/kg) to adult rats. At intervals between 1 and 42 days posttreatment, rats were killed, and the blood levels of FSH, LH, and testosterone were determined together with the levels of immunoactive inhibin in plasma and testicular interstitial fluid (IF). At the same time intervals, seminiferous tubules (ST; 5 x 2 cm) were isolated from control and MAA-treated rats and cultured for 24-72 h in the presence or absence of rat FSH, (Bu)2cAMP, or MAA under rigorously optimized conditions. The hormonal changes observed were related to the presence/absence of specific germ cell types, as determined by assessment of testicular morphology in perfusion-fixed testes from similarly treated rats. One to 3 days after MAA treatment, coincident with the depletion of pachytene spermatocytes, blood levels of FSH were increased significantly compared with controls; FSH returned to control levels at 7-14 days (when early spermatids were depleted), but were increased again at 21-35 days (when late spermatids were depleted). In contrast, while the plasma levels of immunoactive inhibin were increased 2-fold 3 days posttreatment, they were comparable to controls at 7-14 days, but were decreased substantially at 21-28 days. The levels of immunoactive inhibin in testicular IF were more than doubled 1 and 3 days posttreatment, but were comparable to control levels at all other times. Blood levels of LH showed a similar pattern of change to FSH, although only at 21-28 days after MAA treatment was there a significant increase, while blood levels of testosterone were comparable at all times in control and MAA-treated rats. To confirm that the changes observed in vivo after MAA treatment were indicative of changes in Sertoli rather than Leydig cell secretion of immonoactive inhibin, its secretion by isolated ST was assessed, and a pattern of change similar to that in plasma was observed. Thus, when cultured for 24 h under basal conditions, ST from rats 1-3 days after MAA treatment showed a 2- to 3-fold increase in secretion of immunoactive inhibin, which returned to control levels at 7-14 days before being reduced substantially at 21-28 days and then recovering to control levels; similar changes were observed for FSH- and (Bu)2cAMP-stimulated secretion of immunoactive inhibin.(ABSTRACT TRUNCATED AT 400 WORDS)

Background Quantitative Real Time RT-PCR (q2(RT)PCR) is a maturing technique which gives researchers the ability to quantify and compare very small amounts of nucleic acids. Primer design and optimization is an essential yet time consuming aspect of using q2(RT)PCR. In this paper we describe the design and empirical optimization of primers to amplify and quantify plastid RNAs from Zea mays that are robust enough to use with other closely related species. Results Primers were designed and successfully optimized for 57 of the 104 reported genes in the maize plastome plus two nuclear genes. All 59 primer pairs produced single amplicons after end-point reverse transcriptase polymerase chain reactions (RT-PCR) as visualized on agarose gels and subsequently verified by q2(RT)PCR. Primer pairs were divided into several categories based on the optimization requirements or the uniqueness of the target gene. An in silico test suggested the majority of the primer sets should work with other members of the Poaceae family. An in vitro test of the primer set on two unsequenced species (Panicum virgatum and Miscanthus sinensis) supported this assumption by successfully producing single amplicons for each primer pair. Conclusion Due to the highly conserved chloroplast genome in plant families it is possible to utilize primer pairs designed against one genomic sequence to detect the presence and abundance of plastid genes or transcripts from genomes that have yet to be sequenced. Analysis of steady state transcription of vital system genes is a necessary requirement to comprehensively elucidate gene expression in any organism. The primer pairs reported in this paper were designed for q2(RT)PCR of maize chloroplast genes but should be useful for other members of the Poaceae family. Both in silico and in vitro data are presented to support this assumption.

To elucidate the endocrine and paracrine regulation of testicular inhibin production, the effects of follicle-stimulating hormone (FSH), (Bu)2cAMP, germ cells (either crude or enriched preparations) and germ cell-conditioned media on inhibin production (immuno- and bio-activities) and the levels of alpha- and beta B-subunit mRNAs were assessed in cultured Sertoli cells isolated from 20-day-old rats. FSH and (Bu)2-cAMP stimulated both secreted and intracellular inhibin levels in a dose-dependent manner. Using cDNA probes corresponding to the alpha-subunit and the beta B-subunit of rat inhibin it was also shown that both FSH and (Bu)2cAMP markedly increased the level of alpha-subunit mRNA but had no effect on the beta B-subunit mRNA. Addition of a crude mixture of germ cells to Sertoli cell monolayers was found to enhance inhibin secretion. Of the different germ cell fractions tested in co-culture, early spermatids reproducibly stimulated both basal and (Bu)2cAMP-induced production of inhibin whereas pachytene spermatocytes only increased the latter; cytoplasts from elongated spermatids (CES) had no effect. Co-culture of Sertoli cells with liver epithelial cells (LEC) significantly enhanced (Bu)2cAMP-induced inhibin levels. Media conditioned by early spermatids consistently and dramatically stimulated the secretion of both bioactive and immunoactive inhibin by Sertoli cells while spent media from pachytene spermatocytes displayed less activity. CES-conditioned media had only minor stimulatory effects, which may have resulted from the contamination of this fraction by spermatids. Media conditioned by LEC had no effect on inhibin production, confirming that the activity of this cell line is not mediated via a diffusible factor. Early spermatids were found to increase levels of the alpha-subunit mRNA. The current study provides evidence for the involvement of germ cells, in particular of early spermatids, in the local testicular regulation of inhibin gene expression and production in the rat. This may be of crucial importance for the ontogeny of this parameter of Sertoli cell function, and has important implications with regard to the postulated endocrine and paracrine roles of inhibin.

The difference between men and women could be getting smaller, according to a paper that describes a function for female hormones (oestrogens) in the male reproductive tract. Mice that lack the gene for the a form of the oestrogen receptor are infertile, and this turns out to be due to a defect in the process by which sperm are prepared for storage in the testis. Usually, storage involves resorption of fluid from around the sperm, but, in the knockout mice, more fluid is secreted. So the effects of female hormones in males may actually be widespread.

One month after the induction of cryptorchidism in adult rats, serum levels of LH and FSH were significantly elevated in comparison with sham-operated controls, whereas serum levels of testosterone remained low to normal. Testis weight in cryptorchid rats was reduced by over 66%, and once the extratubular fluid was removed by decapsulation, the reduction in weight was 78%. The basal production of testosterone, pregnenolone, and estradiol in vitro by testes from cryptorchid rats was similar to controls, whereas significantly less androstenedione was produced. Testicular stimulation in vitro with a high dose of hCG (360 pM) resulted in significantly greater production of testosterone, pregnenolone, and estradiol by cryptorchid than by control rat tissue. The in vitro binding of [125I]hCG per testis was decreased in the cryptorchid state to 40% of control values, probably as a result of down-regulation of LH receptors due to the 4-fold elevation of serum LH levels in the cryptorchid rats.

Leydig cells were purified on discontinuous Percoll gradients after collagenase digestion of human or rat testes. Leydig cells from both species were found in three bands. As determined by positive staining for 3 beta-hydroxysteroid dehydrogenase, band 1 (lowest density cells) from both species contained only 12-28% Leydig cells. However, while band 3 was the most Leydig cell-enriched fraction in rat cell preparations (70-90% Leydig cells), human band 2 (48-70% Leydig cells) was consistently more Leydig cell enriched than was band 3 (30-56% Leydig cells). Despite their slightly different fractionation pattern, Leydig cells prepared from five men with prostatic carcinoma were similar to those from the rat, both in terms of the amount of testosterone produced basally per 10(6) Leydig cells (80-234 ng/20 h) and in terms of the magnitude of their response to hCG (764-1342 ng/10(6) Leydig cells X 20 h; 5- to 17-fold stimulation above basal). Cells prepared from five other men with prostatic carcinoma produced much lower amounts of testosterone, but still had up to a 6-fold response to hCG. Plasma LH, FSH, and testosterone concentrations in the latter group could not be distinguished from those in the group whose Leydig cells produced large amounts of testosterone in vitro. Morphologically, the testes from the latter group appeared to contain more darkly staining than lightly staining Leydig cells than did the former group. Rat Leydig cells responded in a dose-dependent fashion to hCG over the range 0.03-0.5 mU/mL, whereas human Leydig cells were 10- to 100-fold less sensitive, responding to hCG in the range 0.4-100 mU/mL. The number and affinity of Leydig cell LH (hCG) receptors were assessed from Scatchard analysis of [125I]hCG binding. Compared with rat cells, human Leydig cells contained approximately 20% of the number of LH receptors, while the affinity of the receptors (Kd, approximately 10(-10) M) was similar to that in rats. In conclusion, a method for the isolation of highly responsive human Leydig cells has been developed. The results so far suggest that the function of human Leydig cells may be more similar to that of the rat than thought previously.

Short-term (4 h) incubation of collagenase-dispersed Leydig cells from adult rats in the presence of an LHRH agonist caused a 2-3-fold stimulation (P less than 0.001) of testosterone production. This effect was dose-dependent and as little as 5 x 10(-11) M LHRH agonist caused significant stimulation whilst maximal effects were achieved with 10(-9) M concentrations. Stimulation of steroidogenesis by LHRH agonist was prevented by addition of an antiserum specific for the peptide, but was exaggerated in the presence of the phosphodiesterase inhibitor MIX, suggesting the involvement of cyclic AMP in the response of the Leydig cells to the agonist. Native LHRH caused a similar degree of stimulation of testosterone secretion to LHRH agonist but concentrations 1000 times greater than those of the agonist were required to achieve this, a finding consistent with the known affinities of these 2 peptides for the Leydig cell LHRH-receptor. The addition of LHRH agonist also enhanced (P less than 0.001) testosterone secretion by adult rat Leydig cells in response to hCG or dibutyryl cyclic AMP, and this effect was still evident in the presence of maximally-stimulating concentrations of these factors. LHRH agonist also stimulated testosterone secretion by Leydig cells from immature rats, but this effect differed from that in the adult in being of smaller magnitude and being restricted to effects on basal secretion or secretion elicited by low concentrations of hCG. These results show for the first time (a) that LHRH and its agonists can exert effects on Leydig cell steroidogenesis during short-term incubation, and (b) that these effects are stimulatory, which contrasts with the inhibitory effects reported after long-term (2-3 days) exposure of Leydig cells to LHRH agonists in vivo and in vitro. The availability of this simple and rapid measure of a biological action of LHRH on the Leydig cell should enable its precise mode of action to be determined, and should throw light on the physiological role of endogenously produced testicular 'LHRH'.

Although the testis is under overall control by pituitary gonadotropins, intratesticular control mechanisms are important 1) because of the unique structural organization of the testis, and 2) because of the organization and local requirements of spermatogenesis. The avascular nature of the seminiferous tubules, which comprise 90% of testicular mass in most mammals, means that their exceptionally high energy and nutritional demand has to be met by local transport in testicular interstitial fluid (IF). The rate of formation of IF is determined by capillary permeability and local control of this process is thus a prerequisite for ensuring full nutritional and hormonal support for spermatogenesis. The latter is organized into specific stages arranged in a cycle, each stage having different requirements which only local control mechanisms can ensure. In the rat, Stage VII of the spermatogenic cycle has an absolute requirement for high levels of testosterone and maintenance of such levels in the testicular IF surrounding the tubules thus constitutes a crucially important function requiring local control. Because in this situation, testosterone works via the Sertoli cell and because the testosterone is produced locally by the Leydig cells in response to luteinizing hormone (LH), it is suggested that local control of intratesticular levels of testosterone is likely to be effected by a factor (or factors) produced by the Sertoli cell which acts on the Leydig cell, and which interacts with LH to modulate the levels of testosterone in testicular IF. The possibility that “testicular luteinizing hormone releasing hormone (LHRH)” may fulfill this role is examined in detail by investigating the local effects of an LHRH agonist (LHRH-A) on testicular capillary permeability and the IF levels of testosterone in normal intact adult rats. The results show that LHRH-A has dose-dependent effec-ts on capillary permeability and the IF levels of testosterone and that these effects are modulated according to the ambient level of LH. Alteration of the IF levels of testosterone by LHRH-A is shown to be biologically effective and to be mediated partly by direct effects on Leydig cell steroidogenesis and partly by the control of capillary permeability. The physiological implications and operation of this local mechanism are discussed.

This study was designed to explore the relationship between the intratesticular distribution of testosterone and spermatogenesis by completely destroying the Leydig cells of mature male rats with injection of a single i.p. dose of ethane dimethanesulphonate. After such treatment, testosterone levels in serum, testicular interstitial fluid, seminiferous tubules, and whole testis declined significantly 6 to 24 hours after injection and fell below assay detection limits between 3 and 7 days. At 3 and 7 days, serum LH and FSH levels rose significantly and remained elevated up to 4 and 6 weeks, respectively, in comparison with vehicle-treated controls. Leydig cells disappeared from the interstitium by day 3, but between 2 and 4 weeks postinjection a new generation of fetal-like Leydig cells repopulated the testicular interstitium and, during weeks 6 to 10, were transformed into, or replaced by, Leydig cells with an adult type of morphology. Histologic examination of the seminiferous tubules showed progressive disruption of spermatogenesis between 3 and 14 days post-ethane dimethanesulphonate. The first histologic sign of spermatogenic damage was noted at day 3, with the occurrence of stage-specific degenerating pachytene primary spermatocytes at stages VII to VIII of the spermatogenic cycle. On day 7, these cells and degenerating round, or step 19, spermatids often were observed during stages VII to XI, although qualitatively normal spermatogenesis also was seen in these and all other stages of the cycle. Maximum impairment of spermatogenesis occurred 2 weeks post-ethane dimethane sulphonate, at which time the tubules commonly lacked one or more germ cell generations or, alternatively, showed accumulation of lipid inclusions, extracellular spaces, and variable numbers of degenerating germ cells. Following repopulation of the testis by Leydig cells during weeks 3 and 4, spermatogenesis recovered. By 10 weeks after treatment, qualitatively normal spermatogenesis was seen in the great majority of seminiferous tubules, although a few tubules still remained in which the germ cell complement was severely reduced, and contained only Sertoli cells and spermatogonia.(ABSTRACT TRUNCATED AT 400 WORDS)

The authors recently have reported the presence of a nongonadotropic polypeptide factor in rat testicular interstitial fluid that can exert marked stimulatory effects on Leydig cell testosterone production. To assess the potential physiologic significance of this factor, its effective levels in rat interstitial fluid have been investigated in response to treatments that either markedly reduce interstitial fluid testosterone concentrations (anti-LH treatment; transient or chronic experimental cryptorchidism; destruction of Leydig cells with ethane dimethanesulphonate) or that significantly elevate testosterone levels in interstitial fluid by injection of hCG. The possible relationship between this factor and changes in testicular weight, serum LH and FSH, and interstitial fluid volume also were monitored. When testosterone levels in interstitial fluid were decreased by 75 to 99% either acutely (5-72 hours) or chronically (20-75 days), there was an accompanying increase (P less than 0.001) in the levels of the interstitial fluid factor(s), as determined by the ability of charcoal-stripped interstitial fluid from individual rats to enhance hCG-stimulated testosterone production by Percoll-purified Leydig cells in vitro. Anti-LH treatment increased the levels of the interstitial fluid factor(s) over the ensuing 5 to 48 hours. In abdominal testes from rats made unilaterally or bilaterally cryptorchid for 20 or 55 days, a decrease in interstitial fluid testosterone levels was associated with increased levels of the interstitial fluid factor(s). The same inverse relationship was found 72 hours after treatment with ethane dimethanesulphonate in which Leydig cells had disappeared from the testis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult rats were made unilaterally cryptorchid (UCD) and 6–7 weeks later Leydig cells were isolated from the scrotal and abdominal testes and their capacity to secrete testosterone in vitro was compared. Basal testosterone production by Leydig cells from the abdominal testes of UCD rats was lowered, compared with cells from the contralateral scrotal testes, whilst their responsiveness to both human chorionic gonadotrophin and an LH releasing hormone agonist was enhanced two- to threefold (P< 0·001) compared both with cells from the contralateral scrotal testes and with cells isolated from untreated rats of the same age. In the UCD rats, concentrations of testosterone in testicular interstitial fluid (IF) were reduced (P< 0·001) by 70–90% in abdominal, compared with scrotal, testes. A similar reduction was evident in the levels of testosterone in spermatic venous blood, and both this decrease and that in IF levels of testosterone varied according to the degree of testicular involution. The ontogeny of the above changes was investigated. After induction of unilateral cryptorchidism, the weight of the abdominal compared with the scrotal testis declined slowly, such that by day 5 there was only a 25% reduction in weight compared with a 70% reduction by day 40. In contrast, the levels of testosterone in IF from abdominal testes declined rapidly, such that by day 5 an 80% reduction was attained, compared with scrotal testes, with little further change by day 40. Hormone-stimulated testosterone production by Leydig cells isolated from the abdominal testes was unchanged or marginally reduced over the first 3 days compared with cells from the scrotal testes, but by day 5 there was a significant increase in responsiveness; this increase was of smaller magnitude than that evident at day 40. These results suggest a possible association between the fall in intratesticular levels of testosterone induced by unilateral cryptorchidism and the Leydig cell hypertrophy and hyper-responsiveness that occurs in the same testes. The implications with respect to altered Sertoli–Leydig cell interaction are discussed. J. Endocr. (1984) 102, 319–327

Pituitary-ovarian relationships were studied in seven lactating women by measuring the basal plasma concentrations of pituitary and ovarian hormones and their responses to an estrogen provocation test at 7, 30, and 100 days after delivery. The results were compared to a similar group of seven women who did not breast feed. The first ovulation occurred in five of the nonlactating women beteen 43-87 days after delivery, as judged by the urinary excretion of total estrogen and pregnanediol. In all lactating women, ovarian cyclicity was suppressed for at least 150 days after delivery or until weaning. The basal concentration of PRL in lactating women was significantly higher than in the nonlactating women at all three times measured. At 30 and 100 days, the concentration of estradiol was significantly lower in the lactating women, although the basal concentrations of FSH were similar in the two groups. After an injection of 1 mg estradiol benzoate, the concentrations of FSH and LH in plasma were suppressed to a greater extent in lactating than in nonlactating women. In addition, fewer of the lactating group (one of seven and none of seven at 30 and 100 days, respectively) than the nonlactating group (two of seven and five of seven) subsequently showed a rise in the concentration of LH 58--96 h after the estrogen injection (positive feedback). These results suggest that during lactation the hypothalamic-pituitary system is more sensitive to the negative feedback and relatively insensitive to the positive feedback of estrogen.Pituitary-ovarian relationships were studied in 7 lactating women by measuring the basal plasma concentrations of pituitary and ovarian hormones and their responses to an estrogen provocation test at 7, 30, and 100 days following delivery. The results were compared to a similar group of women who did not breastfeed. The 1st ovulation occurred in 5 of the nonlactating women between 43-87 days after delivery, as judged by the urinary excretion of total estrogen and (PRL) pregnanediol. In all lactating women, ovarian cyclicity was suppressed for at least 150 days after delivery or until weaning. The basal concentration of PRL was significantly higher in lactating women than in the nonlactating at all 3 times measured. At 30 and 100 days, the concentration of estradiol was significantly lower in the lactating women although the basal concentrations of (FSH) follicle stimulating hormone were similar in the 2 groups. After an injection of 1 mg estradiol benzoate, the concentrations of FSH and (LH) luteinizing hormone in plasma were suppressed to a greater extent in lactating than in nonlactating women. In addition, fewer of the lactating women (l of 7 and none of the 7 at 30 and 100 days) than the nonlactating group (2 of 7 and 5 of 7) subsequently showed a rise in the concentration of LH 58-96 hours after the estrogen injection (positive feedback). These results suggest that during lactation the hypothalamic-pituitary system is more sensitive to the negative feedback and relatively insensitive to the positive feedback effect of estrogen.

Immunoreactive inhibin was measured in testicular interstitial fluid (IF) from rats during sexual maturation or after impairment of spermatogenesis induced by ethane dimethanesulphonate (EDS), unilateral cryptorchidism or local heating (43 °C, 30 min) of the testes, to ascertain its usefulness as a marker of changing Sertoli cell function. Cultures of isolated seminiferous tubules were also studied. Inhibin was measured by a radioimmunoassay directed towards the first 26 amino acids of the N-terminus of the α-subunit, and the results confirmed for selected pools of IF by in-vitro bioassay using dispersed ovine pituitary cells. During puberty, IF levels of immunoactive inhibin fell by more than 90% (PPP These results suggest that (1) IF levels of immunoactive inhibin do not always change in parallel to the levels of ABP and may be a useful marker of changing Sertoli cell function, and (2) in at least two situations (puberty and after EDS treatment), there may be a positive relationship between the serum levels of FSH and the IF levels of immunoactive inhibin. This positive relationship was confirmed by in-vitro findings in which FSH and dibutyryl cyclic AMP (but not testosterone) were shown to stimulate immunoactive inhibin production by isolated rat seminiferous tubules during culture for 2–6 days. J. Endocr. (1988) 119, 315–326

To assess the role of androgen feedback in the regulation of the Leydig cell, adult rats were actively immunized against testosterone (T) for a period of 12 months, and Leydig cell function then assessed. In T-immunized rats, the number of LH receptors per Leydig cell and the capacity of these cells to secrete testosterone in vitro were increased 3-fold, while there was a smaller increase in the number of LHRH receptors per cell. These changes were associated with increases in serum levels of LH, FSH, and testosterone of approximately 8-fold, 2.5-fold, and 50-fold, respectively. To test whether increased exposure to LH was responsible for the observed stimulation of Leydig cell function, the effect of a single injection of hCG was assessed. However, this treatment inhibited Leydig cell function, causing a similar loss in the percentage of LH and LHRH receptors in control and T-immunized rats. While hCG injection reduced T responsiveness of Leydig cells from T-immunized rats, surprisingly, it had the opposite effect in controls; the latter effect was probably a consequence of the age of the animals involved. These findings show that interference with androgen feedback results in stimulation of certain parameters of Leydig cell function. However, the observed changes cannot be explained simply by increased exposure to LH, suggesting that other factors, such as intratesticular androgen feedback mechanisms, are involved.

Blood was obtained at various stages of the menstrual cycle from 201 women fitted with intra-uterine contraceptive devices (IUDs), and plasma levels of hCG were determined by radioimmunoassay. Urine samples were obtained from 117 of these women and tested, before and after sephadex gel filtration, in a haemagglutination inhibition test for pregnancy (Pregnosticon). Plasma hCG was undetectable (less than 25 MLU/ml) in all but one of the 201 women and, in this instance, the hCG-assay appeared to be measuring a midcycle peak of LH, as evidenced by high plasma FSH levels. All unextracted urines gave negative results in the Pregnosticon test but, after extraction, 18 of the 117 urines gave positive reactions, most of these being from women at midcycle or in the luteal phase. We conclude that the IUD does not permit the development of the embryo to a point where it is capable of secreting measurable amounts of hCG. Recent claims to the contrary are probably due to cross-reaction of LH or non-specific interference in the assays used for measuring hCG.

This review has shown that local control of testicular function exists at three different levels. (1) Regulation of the entry of substances into the testis via the operation of selective barriers at the level of the capillary endothelium, myoid layer and Sertoli cell tight junctions, and by control of the rate of interstitial fluid formation which is the medium for transport into and within the testis. These mechanisms permit the creation of specific micro-environments around the Leydig cells and outside of the seminiferous tubules as well as within the tubules, prerequisites for the local interaction and communication summarized below. (2) Regulation of spermatogenesis by co-ordination of changes in hormone responsiveness and function of the Sertoli cells with the requirements of the associated germ cells, this varying according to the stage of the spermatogenic cycle. These changes involve regulation of germ cell multiplication, their differentiation and translocation from the basal to the adluminal compartment of the seminiferous tubule and their release into the lumen. (3) Interaction between the seminiferous tubules and the Leydig cells via various factors. The purpose of this interaction is not certain but in the prepubertal testis it probably co-ordinates development of the tubules and Leydig cells, whilst in the adult testis it probably functions to regulate the steroidogenic responsiveness of the Leydig cells in accordance with the stage-dependent requirements of the adjacent seminiferous tubules. Local control mechanisms within the testis therefore appear to co-ordinate the function of the different cell types in the two testicular compartments and, as this is essential for normal function of the testis, the importance of local factors is self-evident. In each of the three areas there is still only limited information as to the precise operation of the various mechanisms and even less is known about the identity of the co-ordinating agents. But, as most of the available information derives only from the last three years, the gaps in our knowledge are to be expected and it is certain that the next few years will see major advances in this area. If the interpretation of the data offered in this review proves accurate then we are on the threshold of a revolution in our understanding of the control of testicular function, and of spermatogenesis in particular. This can only lead to improved methods for regulation of fertility in the male.

The mammalian testis is under the overall control of pituitary gonadotropins but the utilization of these signals to achieve normal testicular function involves complex local interactions between the Sertoli and germ cells, the Sertoli and peritubular cells, and the Sertoli and Leydig cells as well as local control of the testicular vasculature. These interactions serve two purposes: (1) to coordinate the functions of the three testicular compartments (seminiferous tubules, interstitium and the vasculature); and (2) to control the complex but orderly sequence of events that constitutes the spermatogenic cycle. This process, which involves multiplication, differentiation and translocation of the germ cells is organized into a sequence of stages, each of which is composed of a constant association of germ cells at four or five different stages of development. At each stage of the spermatogenic cycle, different events occur and the function of the Sertoli cells alters, probably in accordance with the changing requirements of the associated germ cells. As yet, our understanding of these many local events is extremely limited, particularly with respect to the identity of the hormones/factors involved in controlling the various processes. Our knowledge of paracrine control mechanisms in the testis is derived mainly from studies of the rat, but as the process of spermatogenesis is essentially the same in most mammals and involves the same sequence of events, then findings in the rat can probably be applied in general, if not in detail, to the human testis; the limited direct information available on the human testis supports this view. As most cases of infertility in men occur despite normal or raised serum gonadotropin levels and are characterized by the production of reduced or normal numbers of sperm, then it seems likely that malfunction of one or more of the intricate paracrine processes within the testis may be involved in the aetiology of idiopathic oligospermia. It is therefore argued that advances in our knowledge of the paracrine control of the testis should have major repercussions on our ability to understand, and eventually treat, idiopathic infertility in men, and also to induce infertility for contraceptive purposes.

Summary Rats were treated with a single high dose of methoxy acetic acid (MAA; 650 mg/kg) specifically to deplete seminiferous tubules of pachytene and later spermatocytes. The impact of this selective depletion on subsequent spermatogenesis, sperm output and fertility was then evaluated at intervals ranging from 3 days to 10 weeks. Cauda epididymal sperm number was reduced progressively beyond 2 weeks post-treatment and reached a nadir at 5–6 weeks (28–34% of control values) before recovering progressively back to control levels at 10 weeks. Sperm motility was reduced significantly at 4–7 weeks post-treatment with a nadir at 6 weeks (35% of control values). Thus, at 5–6 weeks after MAA treatment, motile sperm output was reduced by 82–88%. Despite these changes, there was little evidence for infertility in the majority of treated males during a serial mating trial. Evaluation of seminiferous tubule morphology combined with germ cell counts at stage VII of the spermatogenic cycle confirmed that, initially, MAA induced the specific loss of pachytene and later spermatocytes at all stages other than early to mid stage VII. Maturation depletion of germ cells at later invervals was consistent with the initial effects of MAA, although at 21 days post-treatment a number of unpredicted (? secondary) changes in spermatogenesis were observed. These were (a) a reduction in number of pachytene spermatocytes at late stage VII/early stage VIII, (b) retention of sperm at stages IX-XIV, and (c) increased degeneration of pachytene spermatocytes and round spermatids at stage VII and of secondary spermatocytes at stages XIV-I. Whilst none of these changes was severe, together they probably accounted for the unexpectedly prolonged drop in sperm output. It is concluded that whilst deleterious changes in spermatogenesis may occur secondarily following MAA treatment, for the most part spermatogenesis proceeds normally and fertility is largely maintained despite a massive but transient decrease in sperm output.

About one-third of human breast cancers require hormones for their continued growth1, and endocrine ablation or anti-hormone therapy can cause regression of these tumours. As a consequence, ovariectomy in premenopausal women or administration of an anti-oestrogen (tamoxifen) in postmenopausal women represent major options for treatment of metastatic breast cancer. Alternatively, chronic administration of agonistic analogues of luteinizing hormone-releasing hormone (LHRH)2 causes regression of mammary tumours in experimental animals3–6, and such treatment has shown promise in a small series of premenopausal women with advanced breast cancer7. It has been assumed that these results were achieved by suppressing the pituitary–ovarian axis, as the treatment causes a reduction in circulating levels of gonadal steroids similar to that produced by castration6–8. However, LHRH agonists can exert major effects on tissues other than the pituitary in animals9,10 and in the human11–15. Such findings, coupled with reports of LHRH in human breast milk16 and immunohistochemical evidence for the presence of LHRH-like activity in some human breast tumours17, prompted us to test whether LHRH agonists could have direct antitumour effects. We now report major direct effects of LHRH and its agonists on the growth of breast tumour cells in culture.

The primary function of the testosterone secreted by Leydig cells is the maintenance of spermatogenesis and hence fertility. This action of testosterone is mediated by the Sertoli cells which nourish and support the developing spermatozoa. As normal Sertoli cell function is so critically dependent on normal Leydig cell function, a regulatory influence of the Sertoli cells on the Leydig cells has been suggested. Indeed, follicle-stimulating hormone (FSH), which acts only on Sertoli cells, can also cause profound changes in Leydig cell function, although how this is effected is unknown. We recently hypothesized that the Sertoli cell might be the source of a luteinizing hormone releasing hormone (LHRH)-like factor which we detected in the interstitial fluid surrounding the Leydig cells. As injected LHRH agonists cause impairment of gonadal function and directly inhibit FSH-induced changes in Leydig cell function through specific membrane receptors, this 'LHRH-like' factor has all the correct credentials for the postulated messenger between the Sertoli and Leydig cells. Here, we strengthen this case by demonstrating that seminiferous tubules from both the rat and the stumptailed macaque (Macaca arctoides) contain a factor which has LHRH-like receptor-binding and biological activity in vitro, but which is immunologically distinct from native LHRH. We have also shown that this factor is secreted in vitro by cultured rat Sertoli cells.

THE negative feedback action of testicular steroids on circulating levels of luteinising hormone (LH) is well known. It is less clear how the testis influences follicle-stimulating hormone (FSH). The existence of ‘inhibin’, a water-soluble testicular product that inhibits the secretion of FSH, was first postulated in 1932 (ref. 1); recently, inhibin activity has been detected in rete testis fluid2, testicular tissue3, spermatozoa4 and seminal plasma5. Immunisation of rabbits with the inhibin fraction of bull seminal plasma has produced an antiserum which raised plasma FSH levels when injected into male and female rats6. It has been suggested7 that, in the male, inhibin may be produced by the Sertoli cells of the testis. If this is the case, one wonders if inhibin could also be produced by granulosa cells of the ovary. We have, therefore, looked for the presence of a specific FSH-suppressing agent in ovarian follicular fluid by comparing the effects of fluid and peripheral plasma on the levels of FSH and LH in newly castrated male rats.

The stimulatory effects on Leydig cell testosterone secretion of a polypeptide(s) factor present in testicular interstitial fluid (IF) were compared with those of hCG and an LHRH agonist (LHRH-A). The actions of IF and LHRH-A were similar in showing (1) a delayed onset of action, (2) enhancement of testosterone production in response to a maximally stimulating concentration (5 nM) of hCG, and (3) near cessation of stimulation following their removal from the incubation medium. However, addition of an LHRH antagonist blocked only the actions of LHRH-A. Moreover, IF continued to stimulate testosterone production up to at least 20 h either on its own or in the presence of 5 nM hCG, whereas the stimulatory effects of LHRH-A disappeared beyond 6 h. IF was also able to enhance testosterone production in response to LHRH-A or in response to hCG + LHRH-A. IF enhanced testosterone production over 4-20 h in response to all doses of hCG and increasing concentrations of IF caused dose-dependent increments in the rate of hCG (5 nM) stimulated testosterone production. With submaximally stimulating doses of hCG or with LHRH-A alone, the stimulatory effect of IF was more or less additive, whereas with maximally stimulating doses of hCG the effect of IF was clearly synergistic. Thus, whereas the rate of testosterone production by Leydig cells in response to 5 nM hCG declined progressively from 4 to 20 h, addition of IF attenuated or prevented this decline. These findings have implications with respect to the physiological control of intratesticular testosterone levels and with respect to the regulation of steroidogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

This study has evaluated whether the decrease in capacity of Leydig cells to secrete testosterone that occurs during culture or after desensitization with hCG in vivo, is a consequence of the removal of a stimulatory factor(s) in testicular interstitial fluid (IF) to which Leydig cells are normally exposed. When Percoll-purified rat Leydig cells were cultured for 3 days in vitro, there was a progressive reduction in their ability to respond to hCG in terms of either testosterone or progesterone production. In contrast, culture of the cells in the presence of 10% charcoal-stripped IF maintained responsiveness to hCG either partially or completely. This effect was attributable to a factor(s) in IF with a molecular weight of greater than 30 kDa; a comparable fraction from serum had little or no effect. Crude Leydig cells from rats injected 24 h previously with 50 IU hCG showed a 70% reduction in their testosterone response to hCG in vitro and, compared to controls, increased testosterone production poorly in response to increasing concentrations of IF. However, progesterone secretion was increased considerably in response to IF. As in controls, fractionation of crude Leydig cells from hCG-desensitized rats on Percoll gradients resulted in three bands of Leydig cells, except that the yields of band 2 and 3 cells (containing about 40 and 85% Leydig cells, respectively) were reduced by 75% and 90%, respectively, with the majority of Leydig cells remaining in band 1 (which comprises poorly responsive cells). Band 2 and 3 cells from hCG-desensitized rats were not greatly different to cells from control rats in terms of their testosterone response to hCG +/- IF, although they produced considerably more progesterone. It is concluded that the reduced capacity of Leydig cells to secrete testosterone during culture may be attributable to some extent to the removal of a factor(s) in IF to which the cells are normally exposed. In contrast, the reduced in vivo exposure of Leydig cells to such factors, as occurs after hCG injection, cannot explain the poor testosterone response of these cells when they are isolated and cultured.

Adult male rats were treated with ethane dimethanesulphonate (EDS) to destroy the Leydig cells and were then supplemented for 3-10 weeks with testosterone esters (TE) by injection every 3 days. The latter treatment prevented Leydig cell regeneration but maintained quantitatively the androgen-dependent aspects of spermatogenesis, as judged by germ cell counts at stage VII of the spermatogenic cycle. Other than the absence of Leydig cells, the testes of EDS-treated, TE-supplemented rats showed only two morphological changes, (1) the appearance of mast cells throughout the interstitium, and (2) a 3- to 4-fold increase in the number of degenerating germ cells (secondary spermatocytes) at stages XIV-I; this was reflected in a significant decrease in the ratio of spermatids to pachytene spermatocytes at stage VII. These changes were not observed in either oil-treated or TE-treated control rats although similar, but less marked, changes in cell degeneration at stages XIV-I were observed in rats actively immunized against oxytocin. Epididymal sperm number was reduced marginally (approximately 15%) in EDS-treated, TE-supplemented rats while sperm motility was affected even less. In a serial mating trial, some of these treated rats showed evidence of subfertility/infertility, but this was mostly transient and may have been the result of epididymal effects of EDS. These results suggest that Leydig cell products other than testosterone are not essential for maintenance of spermatogenesis and fertility in rats, although because of increased germ cell degeneration during the final stages of meiosis (perhaps as the result of oxytocin withdrawal), a small reduction in sperm count may occur.