Diversity and stability of cultured vaginal lactobacilli in pregnant women from a multi-ethnic urban UK population.

AIM: To determine the diversity and stability of cultured vaginal lactobacilli in a multi-ethnic population of pregnant women. METHODS AND RESULTS: A single-centre, prospective, cohort study was performed in a tertiary perinatal centre in East London, UK. Self-collected vaginal swabs at 13 and 20 weeks gestation were obtained from women attending for routine antenatal care and cultured for lactobacilli. In women who provided both swabs, 37 of 203 (18%) had no lactobacilli cultured at either time. Only 53 (26%) had the same species at both times. Black women were less likely to have lactobacilli cultured at 13 weeks (P = 0·014), and Black and Asian women were less likely to have lactobacilli cultured at 20 weeks (P = 0·002) compared with those in the White and Other groups. CONCLUSIONS: Significant differences exist between ethnic groups in the carriage and stability of vaginal lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: These differences have implications for the design of interventions aimed at normalizing the vaginal microbiota in pregnant women.

Intravenous antibiotics in preterm infants have a negative effect upon microbiome development throughout preterm life.

BACKGROUND: Intestinal dysbiosis is implicated in the origins of necrotising enterocolitis and late-onset sepsis in preterm babies. However, the effect of modulators of bacterial growth (e.g. antibiotics) upon the developing microbiome is not well-characterised. In this prospectively-recruited, retrospectively-classified, case-control study, high-throughput 16S rRNA gene sequencing was combined with contemporaneous clinical data collection, to assess the within-subject relationship between antibiotic administration and microbiome development, in comparison to preterm infants with minimal antibiotic exposure. RESULTS: During courses of antibiotics, diversity progression fell in comparison to that seen outside periods of antibiotic use (-0.71units/week vs. + 0.63units/week, p < 0.01); Enterobacteriaceae relative abundance progression conversely rose (+ 10.6%/week vs. -8.9%/week, p < 0.01). After antibiotic cessation, diversity progression remained suppressed (+ 0.2units/week, p = 0.02). CONCLUSIONS: Antibiotic use has an acute and longer-lasting impact on the developing preterm intestinal microbiome. This has clinical implications with regard to the contribution of antibiotic use to evolving dysbiosis, and affects the interpretation of existing microbiome studies where this effect modulator is rarely accounted for.

Perinatal inflammation influences but does not arrest rapid immune development in preterm babies.

Infection and infection-related complications are important causes of death and morbidity following preterm birth. Despite this risk, there is limited understanding of the development of the immune system in those born prematurely, and of how this development is influenced by perinatal factors. Here we prospectively and longitudinally follow a cohort of babies born before 32 weeks of gestation. We demonstrate that preterm babies, including those born extremely prematurely (<28 weeks), are capable of rapidly acquiring some adult levels of immune functionality, in which immune maturation occurs independently of the developing heterogeneous microbiome. By contrast, we observe a reduced percentage of CXCL8-producing T cells, but comparable levels of TNF-producing T cells, from babies exposed to in utero or postnatal infection, which precedes an unstable post-natal clinical course. These data show that rapid immune development is possible in preterm babies, but distinct identifiable differences in functionality may predict subsequent infection mediated outcomes.

Molecular diagnosis of vascular access device-associated infection in children being treated for cancer or leukaemia.

Blood samples were collected for quantitative 16S rDNA analysis from the vascular access device (VAD) of patients presenting with fever at participating centres of the UK Children's Cancer and Leukaemia Group. In total, 260 of 301 episodes of fever were evaluable and were classified as probable, possible, unlikely or unclassifiable VAD-associated infection. The sensitivity of the 16S rDNA assay declined concomitantly with delays from time of presentation to sampling. The sensitivity with >0.125 pg of bacterial DNA/microL of whole blood was 80% for the 20 probable VAD-associated infections diagnosed with samples collected on the day of or day following presentation. The specificity rose with increasing amounts of bacterial DNA, from 93% with >0.125 pg, to 98% with 0.25-0.5 pg, and to 100% with >0.5 pg/microL blood. The positive predictive value (for probable or possible) was 88% (95% CI 70-98%) with 0.25 pg/microL, and 100% (95% CI 83-100%) with >0.5 pg/microL. All 18 (6.8%) episodes with >0.5 pg of bacterial DNA/microL blood were associated with positive blood cultures. Identifications derived from the DNA sequence were consistent with the blood culture identifications for 15 of the 17 episodes with a DNA sequence identification. The VAD was removed because of suspected infection in six (2.8%) of 216 episodes with <0.125 pg of bacterial DNA/microL, in one (5%) of 20 episodes with 0.125-0.25 pg/microL, in one (16.7%) of six episodes with 0.25-0.5 pg/microL, and in nine (50%) of 18 episodes with >0.5 pg/microL. A bacterial DNA concentration of >0.5 pg/microL in blood drawn through a central venous catheter at the time of fever presentation had a high positive predictive value for VAD-associated infection and predicted an increased risk of VAD removal because of suspected infection.

Immunohistochemical localization of androgen receptors in the rat testis: evidence for stage-dependent expression and regulation by androgens.

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.

Comparative effects of neonatal exposure of male rats to potent and weak (environmental) estrogens on spermatogenesis at puberty and the relationship to adult testis size and fertility: evidence for stimulatory effects of low estrogen levels.

This study investigated whether neonatal exposure of male rats to estrogenic compounds altered pubertal spermatogenesis (days 18 and 25) and whether the changes observed resulted in long-term changes in testis size, mating, or fertility (days 90-100). Rats were treated neonatally with a range of doses (0.01-10 microg) of diethylstilbestrol (DES; administered on alternate days from days 2-12), a high dose of octylphenol (OP; 2 mg administered daily from days 2-12) or bisphenol A (Bis-A; 0.5 mg administered daily from days 2-12), or vehicle, while maintained on a standard soy-containing diet. The effect on the same parameters of rearing control animals on a soy-free diet was also assessed as was the effect of administering such animals genistein (4 mg/kg/day daily from days 2-18). Testis weight, seminiferous tubule lumen formation, the germ cell apoptotic index (apoptotic/viable germ cell nuclear volume), and spermatocyte nuclear volume per unit Sertoli cell nuclear volume were used to characterize pubertal spermatogenesis. Compared with (soy-fed) controls, DES administration caused dose-dependent retardation of pubertal spermatogenesis on day 18, as evidenced by decreases in testis weight, lumen formation, and spermatocyte nuclear volume per unit Sertoli cell and elevation of the germ cell apoptotic index. However, the two lowest doses of DES (0.1 and 0.01 microg) significantly increased spermatocyte nuclear volume per unit Sertoli cell. Similarly, treatment with either OP or Bis-A significantly advanced this and some of the other aspects of pubertal spermatogenesis. Maintenance of control animals on a soy-free diet also significantly advanced lumen formation and spermatocyte nuclear volume per unit Sertoli cell compared with controls fed a soy-containing diet. Administration of genistein reversed the stimulatory effects of a soy-free diet and significantly retarded most measures of pubertal spermatogenesis. In general, plasma FSH levels in the treatment groups changed in parallel to the spermatogenic changes (reduced when pubertal spermatogenesis retarded, increased when pubertal spermatogenesis advanced). By day 25, although the changes in FSH levels largely persisted, all of the stimulatory effects on spermatogenesis seen on day 18 in the various treatment groups were no longer evident. In adulthood, testis weight was decreased dose dependently in rats treated neonatally with DES, but only the lowest dose group (0.01 microg) showed evidence of mating (3 of 6) and normal fertility (3 litters). Animals treated neonatally with OP or Bis-A had normal or increased (Bis-A) testis weights and exhibited reasonably normal mating/fertility. Animals fed a soy-free diet had significantly larger testes than controls fed a soy-containing diet, and this difference was confirmed in a much larger study of more than 24 litters, which also showed a significant decrease in plasma FSH levels and a significant increase in body weight in the males kept on a soy-free diet. Neonatal treatment with genistein did not alter adult testis weight, and although most males exhibited normal mating and fertility, a minority did not mate or were infertile. It is concluded that 1) neonatal exposure of rats to low levels of estrogens can advance the first wave of spermatogenesis at puberty, although it is unclear whether this is due to direct effects of the estrogen or to associated elevation of FSH levels; 2) the effect of high doses of OP and Bis-A on these processes is essentially benign; and 3) the presence or absence of soy or genistein in the diet has significant short-term (pubertal spermatogenesis) and long-term (body weight, testis size, FSH levels, and possibly mating) effects on males.

Permanent effects of neonatal estrogen exposure in rats on reproductive hormone levels, Sertoli cell number, and the efficiency of spermatogenesis in adulthood.

This study aimed to identify the mechanism(s) for impairment of spermatogenesis in adulthood in rats treated neonatally with estrogens. Rats were treated (days 2-12) with 10, 1, or 0.1 microg diethylstilbestrol (DES), 10 microg ethinyl estradiol (EE), 10 mg/kg of a GnRH antagonist (GnRHa), or vehicle and killed in adulthood. DES/EE caused dose-dependent reductions in testis weight, total germ cell volume per testis, and Sertoli cell volume per testis. Sertoli cell number at 18 days of age in DES-treated rats was reduced dose dependently. GnRHa treatment caused changes in these parameters similar to those in rats treated with 10 microg DES. Plasma FSH levels were elevated (P < 0.001) to similar levels in all treatment groups regardless of differences in Sertoli cell number and levels of inhibin B; the latter reflected Sertoli cell number, but levels were disproportionately reduced in animals treated with high doses of DES/EE. Neonatal estrogen treatment, but not GnRHa, caused dose-dependent reductions (40-80%) in plasma testosterone levels in adulthood, but did not alter LH levels. Preliminary evidence suggests that the decrease in testosterone levels in estrogen-treated rats is not due to reduced Leydig cell volume per testis. GnRHa-treated rats exhibited a significant increase in germ cell volume per Sertoli cell and a reduction in germ cell apoptosis, probably because of the raised FSH levels. Despite similar raised FSH levels, rats treated with DES (10 or 1 microg) or EE (10 microg) had reduced germ cell volume/Sertoli cell and increased germ cell apoptosis, especially when compared with GnRHa-treated animals. The latter changes were associated with an increase in lumen size per testis, indicative of impaired fluid resorption from the efferent ducts, resulting in fluid accumulation in the testis. Rats treated neonatally with 0.1 microg DES showed reduced germ cell apoptosis comparable to that in GnRHa-treated animals. The changes in apoptotic rate among treatment groups occurred across all stages of the spermatogenic cycle. It is concluded that 1) neonatal estrogen treatment results in dose-dependent alterations in Sertoli cell numbers, germ cell volume, efficiency of spermatogenesis, and germ cell apoptosis in adulthood; 2) the relatively poor spermatogenesis in estrogen-treated animals is most likely due to altered testis fluid dynamics and/or altered Sertoli cell function; 3) as indicated by FSH (LH) and testosterone levels, the hypothalamic-pituitary axis and Leydig cells are probably more sensitive than the Sertoli cells to reprogramming by estrogens neonatally; and 4) elevated FSH levels in adulthood may improve the efficiency of spermatogenesis.

Age-, cell- and region-specific immunoexpression of estrogen receptor alpha (but not estrogen receptor beta) during postnatal development of the epididymis and vas deferens of the rat and disruption of this pattern by neonatal treatment with diethylstilbestrol.

This study in rats sought to 1) characterize immunoexpression of estrogen receptor alpha (ERalpha) and ERss in the efferent ducts, epididymis, and vas deferens during postnatal development; 2) establish whether ER expression changed after neonatal treatment with diethylstilbestrol (DES); and 3) determine whether ER changes coincided with abnormal epididymal/vas development. Rats were administered 10 microg DES or vehicle on days 2, 4, 6, 8, 10, and 12 and were sampled on days 10, 18, 25, 35, and 90+. At all ages, ERalpha was immunoexpressed intensely in the efferent ducts. On day 10, immunoexpression of ERalpha was absent from the epididymis and vas, but was detectable on day 18 in epithelial cells in the caput, corpus, and proximal cauda. Epithelial expression of ERalpha was absent from the distal cauda and in the proximal and distal vas was confined to a band of periductal stromal cells. Thus, on day 18, the site of ERalpha expression delineated the epididymis-vas boundary. On days 25-35, epithelial expression of ERalpha was absent, but stromal expression persisted in the vas and distal cauda. In adults, immunoexpression of ERalpha in the epididymis and vas was absent. In contrast, ERbeta was immunoexpressed in epithelial cells and some stromal cells in the efferent ducts, epididymis, and vas at all ages. In the vas, stromal expression of ERalpha and ERbeta was in different layers. DES treatment caused 1) underdevelopment of the epididymal duct and reduced epithelial height in epididymis and vas; 2) coiling of the extraepididymal vas; 3) thickening of the periductal actin-free stromal layer in the distal cauda and vas; and 4) reduced cell proliferation on day 18 in the epididymis and vas, based on incorporation of bromodeoxyuridine, especially in the epithelium. These changes coincided with abnormalities in cell- and region-specific immunoexpression of ERalpha, but not ERbeta. Thus, in DES-treated rats on day 18, epithelial expression of ERalpha occurred in all regions of the epididymis and vas instead of being confined to the caput, corpus, and proximal cauda as in controls. Similarly, stromal ERalpha expression in the vas of DES-treated rats was not confined to a periductal layer as in controls, but occurred diffusely in the muscle layer. It is suggested that 1) estrogens play a role in peripubertal development of the epididymis and vas; 2) the cellular site of expression of ERalpha either plays a role in or reflects demarcation of the epididymal/vas boundary; and 3) blurring of this boundary in DES-treated rats coincides with altered ERalpha immunoexpression.

Estrogen receptor beta, but not estrogen receptor alpha, is present in the vascular endothelium of the human and nonhuman primate endometrium.

Estrogen action is dependent upon the presence of specific ligand-activated receptors in target tissues. The aim of the present experiments was to compare the spatial and temporal pattern of expression of estrogen receptor beta (ERbeta) with that of ERalpha in full thickness endometrial samples (from the superficial to the basal zone) obtained from both women and rhesus macaques. Immunohistochemical localization with specific antibodies revealed that ERalpha and ERbeta were both expressed in nuclei of the glands and stroma. Consistent with previous studies, expression of ERalpha declined in the glands and stroma of the functionalis during the secretory phase. The luminal epithelium also displayed positive immunoreactivity for ERbeta. Expression of ERbeta declined in glandular cell nuclei, but not stroma, within the functionalis during the late secretory phase. Levels of expression of ERalpha and ERbeta in all cellular compartments remained unchanged in the basalis. Both receptor subtypes were detected on Western blots using proteins extracted from uterine samples obtained throughout the menstrual cycle. There was a striking contrast between the pattern of expression of ERalpha and ERbeta in the vascular endothelium and the perivascular cells surrounding endometrial blood vessels; only ERbeta was present in the endothelial cell population, although both forms of ER were expressed in perivascular cells. We conclude that estrogen action(s) within the vascular endothelium in the endometrium may be mediated via direct binding to the ERbeta isoform and that these cells could therefore be a target for agonists or antagonists that selectively target the beta form of the ER.

Investigation of the potential role of the germ cell complement in control of the expression of transferrin mRNA in the prepubertal and adult rat testis.

Iron is required for the normal development of germ cells during spermatogenesis. Because these cells have no direct access to systemic iron, there exists a shuttle system involving production and secretion of the iron-transporting protein transferrin by the Sertoli cells. Previous reports using cultures of immature Sertoli cells exposed to adult germ cells, or in vivo studies involving germ cell depleted adult rat testes, concluded that production of transferrin by Sertoli cells is modulated by germ cell complement. In the present study we have used in situ hybridisation with cRNA probes directed against the 5' and 3' ends of transferrin mRNA to examine the pattern of expression of transferrin in the immature and adult rat testis. Adult rats were treated with ethane dimethane sulphonate or methoxyacetic acid (MAA) to manipulate their testosterone levels or germ cell complement respectively. Initial findings obtained using the 3' probe showed a decrease in transferrin mRNA associated with round spermatid depletion. However, these data were not confirmed by in situ hybridisation when the 5' probe was used. The specificity of the probes was examined using Northern blotting and the 3' probe was found to hybridise to the germ cell transcript for hemiferrin even under conditions of high stringency. Examination of immature and pubertal rat testes by in situ hybridisation using the 5' transferrin-specific probe found that as early as 14 days of age the level of expression of transferrin mRNA was clearly different between tubules, and the mRNA appeared to be expressed in Leydig cells on and after day 31. In the adult rat testis, maximal expression of transferrin mRNA was found at stages VIII-XIV, calling into question the interpretation of the results of some previous studies showing expression of transferrin mRNA at all stages of the spermatogenic cycle. This stage-specific pattern of expression was not altered by acute germ cell depletion using MAA. However, Northern blot analysis showed a statistically significant increase in transferrin mRNA expression at 7 days after MAA treatment when pachytene spermatocytes were depleted from tubules at all stages of the spermatogenic cycle at which transferrin is normally expressed. In conclusion, we found that transferrin mRNA expression was not modulated by round spermatids as has been reported previously but that meiotic germ cells may influence expression of transferrin at specific stages of the spermatogenic cycle.

Localization of the sites of synthesis and action of prolactin by immunocytochemistry and in-situ hybridization within the human utero-placental unit.

While the fetal pituitary synthesizes and releases prolactin, it is also produced within the utero-placental unit during pregnancy in women and has been localized in the amnion, chorion and decidua. However, it is not clear whether prolactin is synthesized within all these non-fetal pituitary tissues. We have investigated prolactin production and its gene expression using tissue culture, immunocytochemistry and in-situ hybridization techniques. Prolactin was immunolocalized not only in the decidua but also in amnion and trophoblast cells. In contrast, the in-situ hybridization results showed that silver grains, formed by specific hybridization of a prolactin cDNA probe to prolactin mRNA, were confined to decidual cells of early and term pregnancy. The results from tissue cultures correlated well with those of in-situ hybridization, that is that only the decidua made detectable prolactin, while it was undetectable in the culture medium from trophoblast tissue, irrespective of the stage of pregnancy. This study, for the first time, establishes that only decidualized cells are involved in biosynthesis of prolactin; other prolactin-containing cells in the amnion and trophoblast appear to sequester prolactin, possibly via receptors, suggesting that prolactin may play an important paracrine role within the amnion and syncitio- and cytotrophoblast of the utero-placental unit.

Immunolocalisation of androgen receptor to interstitial cells in fetal rat testes and to mesenchymal and epithelial cells of associated ducts.

Androgens are required for the development of male internal and external genitalia. Androgen action is mediated by an intracellular receptor which acts as a transcription factor following activation by ligand binding. The aim of the present study was to define the time of appearance of androgen receptor (AR) in the male fetal rat gonad using immunohistochemistry. Intact fetuses (days 13.5-16.5) or testicular tissue (days 16.5-20.5 and days 3-7 postnatal) were fixed in Bouins' solution and processed into paraffin wax. On day 16.5 nuclear AR were present in mesenchymal cells surrounding the Wolffian duct but those around the Mullerian duct were receptor negative. During the following day (17-18) the abundance of nuclear staining increased, becoming detectable in the epithelial cells of the Wolffian ducts. Within the testis some nuclear staining was apparent at day 17 but was confined to interstitial cells surrounding the seminiferous cords. As development of the testis proceeded the abundance of nuclear AR in peritubular and elongated mesenchymal cells increased. AR were not detected in fetal Leydig cells expressing 3 beta-hydroxysteroid dehydrogenase nor in the ovaries or associated ducts of female fetuses at the same ages. In conclusion, in the rat we have found AR expression detectable by immunohistochemistry in mesonephric mesenchyme to be confined to that underlying the Wolffian ducts and to be absent from the area around the degenerating Mullerian duct. On and after day 17 of gestation AR is present in Wolffian duct epithelial cell nuclei and within the testis it is confined to peritubular and interstitial cells which may have migrated from the mesonephros.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of oestrogen receptor beta (ER beta) in multiple rat tissues visualised by immunohistochemistry.

Steroid hormones regulate cell function via specific receptors, members of a super family of ligand activated transcription factors, expressed in their target tissues. A second oestrogen receptor (ER beta) has recently been shown by RT-PCR to have a wide tissue distribution distinct from that of oestrogen receptor alpha (ER alpha). We have raised a polyclonal antiserum using a peptide specific for ER beta in order to determine the cellular sites of expression of the receptor. In the adult rat ER beta was localised to cell nuclei in a wide range of tissues including ovary, oviduct, uterus, lung, adrenal, seminal vesicle, bladder, heart, prostate and testis. In the ovary ER beta was present in multiple cell types including granulosa cells in small, medium and large follicles, theca and corpora lutea whereas ER alpha was undetectable in these cell types. In the uterus ER beta and ER alpha were both present in epithelial cells lining the lumen and glands. In the lung ER beta was present in the cells lining the bronchioles and alveoli as well as in smooth muscle. In bladder and seminal vesicle immunostaining was intense in epithelial cells but the receptor was also expressed in nuclei of smooth muscle cells. Cell nuclei of the heart ventricle were immunopositive for ER beta as were most cells of the adult rat adrenal. In the seminiferous epithelium of the testis, nuclei of Sertoli cells were immunopositive but expression was not stage dependent. In conclusion, immunohistochemistry has proved invaluable in visualising specific sites of expression of ER beta in complex tissues including those of the reproductive tract.

Ontogeny of anti-müllerian hormone, 3 beta-hydroxysteroid dehydrogenase and androgen receptor expression during ovine total gonadal development.

Anti-müllerian hormone (AMH) and androgenic steroids are key factors regulating the masculinisation of the internal and external genitalia during fetal development. AMH is produced in Sertoli cells and causes regression of the müllerian ducts in the male. 3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) is one of the key steroidogenic enzymes regulating testosterone production in Leydig cells. The objective of this experiment was to elucidate the development of the ovine fetal testes by identifying the spatio-temporal expression of AMH, 3 beta-HSD and androgen receptor expression within them. Fetuses from days 30 and 40 of gestation were fixed intact, while the gonads were dissected from the fetuses on days 70, 100 and 130 of gestation. Tissue was fixed in Bouin's fixative for 6 h, processed into paraffin wax and sections immunostained using rabbit anti-human AMH, 3 beta-HSD or androgen receptor antibodies. While seminiferous cords were absent on day 30 of gestation, pre-cord organisation was apparent and the gonad could be clearly distinguished from surrounding tissue by the presence of AMH and 3 beta-HSD immunopositive cells. Androgen receptor expression was not apparent at this stage. By day 40 of gestation the testis was organised into distinct seminiferous cords and intense immunostaining for AMH and 3 beta-HSD was present in Sertoli cells within the cords and Leydig cells in the interstitium respectively. Androgen receptor immunopositive cells were present in the interstititum but cells destined to develop into rete testis were immunonegative. By day 70 of gestation, the rete testis was organised in the centre of the testis and was strongly androgen receptor immunopositive. AMH and 3 beta-HSD expression was present in Sertoli and Leydig cells respectively. The expression of AMH, 3 beta-HSD and androgen receptor in the 100 and 130 day gestation fetuses was similar to that identified in the 70 day fetuses. In conclusion, Sertoli and Leydig precursor cells are present in the gonad prior to seminiferous cord formation and contain AMH and 3 beta-HSD at all stages of gestation examined. While androgen receptor immunoexpression was present in nuclei of interstitial cells from day 40 of gestation and in the rete testis from day 70 of gestation, Sertoli cells were immunonegative for androgen receptor at all of the stages examined.

Expression of oestrogen receptor beta (ER beta) occurs in multiple cell types, including some germ cells, in the rat testis.

The identification of a second oestrogen receptor (beta) has prompted a re-evaluation of the potential sites of action of oestrogens. The aim of the present study was to characterize immunoexpression of ER beta expression in the testis to complement earlier data which had demonstrated that expression of ER alpha is confined to testicular interstitial Leydig cells. In all testes studied, including those from both fetal (day 20.5 p.c.) and adult rats, ER beta was found to be expressed in multiple cell types. Sertoli cell nuclei were immunopositive at all ages. In adult testes expression in Sertoli cells was not stage dependent and was unaffected by ablation of Leydig cells. In fetal testes ER beta was also expressed in peritubular cells, fetal Leydig cells and gonocytes. In the pubertal and adult testis ER beta was detected in the nuclei of spermatogonia and most pachytene spermatocytes. Weak immunopositive staining was present in the cytoplasm of spermatocytes undergoing the second meiotic division. In conclusion the widespread expression of ER beta in the testis is consistent with a role for oestrogens in modulating spermatogenesis, and hence fertility, in the male.

Immunocytochemical localization of inhibin alpha-subunit in the human corpus luteum.

The localization of inhibin alpha-subunit within the human corpus luteum was investigated. The antiserum used was raised in sheep against the first 1-23 amino acid sequence of the N-terminus of the human inhibin alpha-subunit. Using the avidin-biotin immunoperoxidase technique, intense immunostaining was localized within the granulosa-lutein cells of the corpus luteum, with absence of staining in the theca-lutein cells and surrounding ovarian tissue. Similar distribution of inhibin alpha-subunit immunostaining was observed in 12 corpora lutea obtained during the early, mid- and late-luteal phases and no changes in intensity were apparent at these different stages. Negative controls were obtained by applying antiserum which had been preabsorbed overnight with excess inhibin peptide in place of primary antiserum and also normal nonimmune sheep serum as a substitute for primary antiserum. These results provide further evidence that the human corpus luteum is a significant source of immunoreactive inhibin during the normal human menstrual cycle. The specific localization within the granulosalutein cells of the corpus luteum suggests that inhibin alpha-subunit production may originate from a discrete cell population within the human corpus luteum.

Developmental regulation of androgen receptor in rat ovary.

Androgen receptor (AR) distribution and developmental regulation in the rat ovary were examined by semiquantitative immunohistochemistry. Ovarian AR mRNA levels were also determined by Northern analysis of total RNA and compared with the levels of cytochrome P450aromatase (P450arom), an established marker of preovulatory follicular maturity. Hypophysectomized immature female rats were treated with recombinant human (rh)-FSH and/or rh-LH, or human menopausal gonadotrophin (HMG). AR was predominantly located in granulosa cells. There was no indication of specific AR immunoreactivity in thecal cells, but scattered stromal cells did stain positively. In control and LH-treated ovaries, only small preantral/early antral follicles were present. Granulosa cells in these follicles showed intense AR immunostaining. Treatment with FSH, FSH and LH or HMG stimulated varying degrees of preovulatory follicular development. In these follicles, the intensity of AR immunostaining progressively declined as follicular development progressed. In intact immature rats treated with FSH, the abundance of ovarian AR mRNA was significantly decreased to 35% of the control value while combined treatment of FSH and LH resulted in further down-regulation of AR mRNA expression to 17% of the control value. A decrease in the abundance of AR mRNA was accompanied by a simultaneous increase in the abundance of P450arom mRNA. Similar results were obtained in hypophysectomized immature rats treated with FSH and LH, suggesting an inverse relationship between AR mRNA expression and granulosa cell maturity. These results suggest that (1) the AR is most abundant in the granulosa cells of rat ovaries and (2) the expression of AR and its mRNA are developmentally regulated, being down-regulated during FSH-stimulated preovulatory follicular development.

Immunolocalisation of oestrogen receptor-alpha within the testis and excurrent ducts of the rat and marmoset monkey from perinatal life to adulthood.

The sites of action and the physiological role of oestrogens in the male reproductive tract are poorly understood. We have undertaken a systematic study of the immunoexpression of oestrogen receptor-alpha (ER alpha) in the male rat from late fetal life through to adulthood and compared the findings with results obtained in the marmoset monkey (Callithrix jacchus) from neonatal to adult life. The testes, rete testis, efferent ducts and epididymis were examined from normal male rats (aged 4, 8, 10, 15, 20, 25, 38, 48 and 90 days) and from male rat fetuses on days 17.5 and 18.5 of gestation; comparable tissues were examined from neonatal, infantile, peripubertal and adult marmosets aged 8, 18-24, 54-62 and 92-112 weeks respectively. Immunolocalisation of ER alpha used antigen retrieval and a monoclonal antibody directed to the N-terminus, which had proved superior to six other antisera tested. ER alpha was immunoexpressed in interstitial cells, including the fetal/ neonatal generation of Leydig cells, in both the rat and marmoset. In the rat, the adult generation of Leydig cells were also immunopositive for ER alpha whereas the comparable cells in the marmoset were only weakly immunopositive. ER alpha was not expressed in Sertoli cells, peritubular myoid cells, blood vessels or germ cells at any time in either species. In late fetal life in the rat, ER alpha was immunoexpressed in cells surrounding the mesonephric tubules, whereas postnatally it was expressed in the epithelium of the rete testis and efferent ducts at all ages from 4 to 90 days; this immunoexpression was most pronounced in the efferent ducts. In the marmoset, the efferent ducts, but not the rete testis, also showed intense immunoexpression of ER alpha. Apart from sporadic immunostaining for ER alpha in the epididymal duct of the rat in the neonatal period, the caput, corpus and cauda epididymis were negative for immunoexpression of ER alpha at all ages in both species. These findings suggest that the main actions of oestrogens in the male reproductive tract, mediated by ER alpha, are related to the development and function of the efferent ducts and the Leydig cells. In consideration of data from this and previous studies of oestrogen binding, we predict possible sites of expression of other oestrogen receptors (e.g. ER beta) in Sertoli cells and the epididymis. Interactive effects, related to the relative levels of androgens and oestrogens, could be physiologically important in the excurrent ducts of the adult testis.

Development and validation of a new monoclonal antibody to mammalian aromatase.

The biosynthesis of oestrogens from androgens is catalysed by the aromatase complex, an essential component of which is the aromatase cytochrome P450 (P450 arom) protein. Expression of a functional P450 arom is essential for normal fertility in males and females and the sequence of the protein is highly conserved. We have raised a new monoclonal antibody against a conserved peptide and validated it on fixed tissue sections of the rat, common marmoset (Callthrix jacchus) and human. The monoclonal antibody was used successfully for Western analysis and specifically reacted with a 55 kDa protein in microsomal extracts. On sections of ovaries in all three species, expression in follicles was specific to the mural granulosa cells of antral follicles and was present in corpora lutea. In the human and marmoset, staining of luteal cells was markedly heterogeneous and did not appear to vary consistently with the stage of the cycle. The intensity of immunostaining was elevated in corpora lutea from pregnant rats and following human chorionic gonadotropin rescue in the human. In the testis, the highest levels of expression were observed in the Leydig cells within the interstitium. In adult rat and marmoset, and possibly also in the human, some P450 arom was associated with the cytoplasm surrounding elongate spermatids but other germ cells were immunonegative. In conclusion, a new monoclonal antibody specific for P450 arom recognises the protein in rodent, primate and human. Its ability to work on fixed tissue sections will facilitate identification of individual cells expressing P450 arom within complex tissues.