Ana Monteiro

Spermatogenesis and Sertoli cell activity in mice lacking Sertoli cell receptors for follicle stimulating hormone and androgen

Spermatogenesis in the adult male depends on the action of FSH and androgen. Ablation of either hormone has deleterious effects on Sertoli cell function and the progression of germ cells through spermatogenesis. In this study we generated mice lacking both FSH receptors (FSHRKO) and androgen receptors on the Sertoli cell (SCARKO) to examine how FSH and androgen combine to regulate Sertoli cell function and spermatogenesis. Sertoli cell number in FSHRKO-SCARKO mice was reduced by about 50% but was not significantly different from FSHRKO mice. In contrast, total germ cell number in FSHRKO-SCARKO mice was reduced to 2% of control mice (and 20% of SCARKO mice) due to a failure to progress beyond early meiosis. Measurement of Sertoli cell-specific transcript levels showed that about a third were independent of hormonal action on the Sertoli cell, whereas others were predominantly androgen dependent or showed redundant control by FSH and androgen. Results show that FSH and androgen act through redundant, additive, and synergistic regulation of spermatogenesis and Sertoli cell activity. In addition, the Sertoli cell retains a significant capacity for activity, which is independent of direct hormonal regulation.

Up-regulation of metabotropic glutamate receptor subtypes 3 and 5 in spinal cord in a clinical model of persistent inflammation and hyperalgesia

&NA; Evidence from experimental pain research has revealed that metabotropic glutamate receptors (mGluRs) play a pivotal role in nociceptive processing, inflammatory pain and hyperalgesia. The aim of this study was to characterise expression of group I and II mGluRs in spinal cord in a model of naturally occurring persistent inflammation (sheep with unilateral lameness due to inflammation of the digital tissues of the feet, estimated to have been affected by the condition for >2 weeks) and an experimental model of acute inflammation (injection of intradermal carrageenan into lower forelimb in sheep). Animals with unilateral clinical inflammation displayed significant mechanical hyperalgesia on the affected limb. Carrageenan treatment produced significant bilateral limb mechanical hyperalgesia 3 h post‐injection. Up‐regulation of mGluR3 and mGluR5 mRNA was observed in ipsilateral spinal cord recovered from clinically lame animals, restricted to laminae II–V and I–II, respectively. Western blot analyses of protein extracts revealed a bilateral increase in mGluR2/3 and mGluR5. No change was detected in spinal cord mGluR1 or mGluR2 mRNA. There was no change in mGluR1,2,3,5 subtype mRNA or proteins in spinal cord recovered from animals 3 h post‐carrageenan. These results demonstrate for the first time that mGluR subtypes are differentially expressed in spinal cord dorsal horn in response to persistent inflammation, and suggest that mGluR activity may be involved in mediating altered behaviours associated with clinical inflammatory pain.

Development of steroid signaling pathways during primordial follicle formation in the human fetal ovary.

CONTEXT Ovarian primordial follicle formation is critical for subsequent human female fertility. It is likely that steroid, and especially estrogen, signaling is required for this process, but details of the pathways involved are currently lacking. OBJECTIVE The aim was to identify and characterize key members of the steroid-signaling pathway expressed in the second trimester human fetal ovary. DESIGN We conducted an observational study of the female fetus, quantifying and localizing steroid-signaling pathway members. SETTING The study was conducted at the Universities of Aberdeen, Edinburgh, and Glasgow. PATIENTS/PARTICIPANTS Ovaries were collected from 43 morphologically normal human female fetuses from women undergoing elective termination of second trimester pregnancies. MAIN OUTCOME MEASURES We measured mRNA transcript levels and immunolocalized key steroidogenic enzymes and steroid receptors, including those encoded by ESR2, AR, and CYP19A1. RESULTS Levels of mRNA encoding the steroidogenic apparatus and steroid receptors increased across the second trimester. CYP19A1 transcript increased 4.7-fold during this period with intense immunostaining for CYP19A detected in pregranulosa cells around primordial follicles and somatic cells around oocyte nests. ESR2 was localized primarily to germ cells, but androgen receptor was exclusively expressed in somatic cells. CYP17A1 and HSD3B2 were also localized to oocytes, whereas CYP11A1 was detected in oocytes and some pregranulosa cells. CONCLUSIONS The human fetal ovary expresses the machinery to produce and detect multiple steroid signaling pathways, including estrogenic signaling, with the oocyte acting as a key component. This study provides a step-change in our understanding of local dynamics of steroid hormone signaling during the key period of human primordial follicle formation.

Effect of FSH on testicular morphology and spermatogenesis in gonadotrophin-deficient hypogonadal mice lacking androgen receptors.

FSH and androgen act to stimulate and maintain spermatogenesis. FSH acts directly on the Sertoli cells to stimulate germ cell number and acts indirectly to increase androgen production by the Leydig cells. In order to differentiate between the direct effects of FSH on spermatogenesis and those mediated indirectly through androgen action, we have crossed hypogonadal (hpg) mice, which lack gonadotrophins, with mice lacking androgen receptors (AR) either ubiquitously (ARKO) or specifically on the Sertoli cells (SCARKO). These hpg.ARKO and hpg.SCARKO mice were treated with recombinant FSH for 7 days and testicular morphology and cell numbers were assessed. In untreated hpg and hpg.SCARKO mice, germ cell development was limited and did not progress beyond the pachytene stage. In hpg.ARKO mice, testes were smaller with fewer Sertoli cells and germ cells compared to hpg mice. Treatment with FSH had no effect on Sertoli cell number but significantly increased germ cell numbers in all groups. In hpg mice, FSH increased the numbers of spermatogonia and spermatocytes, and induced round spermatid formation. In hpg.SCARKO and hpg.ARKO mice, in contrast, only spermatogonial and spermatocyte numbers were increased with no formation of spermatids. Leydig cell numbers were increased by FSH in hpg and hpg.SCARKO mice but not in hpg.ARKO mice. Results show that in rodents 1) FSH acts to stimulate spermatogenesis through an increase in spermatogonial number and subsequent entry of these cells into meiosis, 2) FSH has no direct effect on the completion of meiosis and 3) FSH effects on Leydig cell number are mediated through interstitial ARs.

Direct Action through the Sertoli Cells Is Essential for Androgen Stimulation of Spermatogenesis

Androgens act to stimulate spermatogenesis through androgen receptors (ARs) on the Sertoli cells and peritubular myoid cells. Specific ablation of the AR in either cell type will cause a severe disruption of spermatogenesis. To determine whether androgens can stimulate spermatogenesis through direct action on the peritubular myoid cells alone or whether action on the Sertoli cells is essential, we crossed hypogonadal (hpg) mice that lack gonadotrophins and intratesticular androgen with mice lacking ARs either ubiquitously (ARKO) or specifically on the Sertoli cells (SCARKO). These hpg.ARKO and hpg.SCARKO mice were treated with testosterone (T) or dihydrotestosterone (DHT) for 7 d and testicular morphology and cell numbers assessed. Androgen treatment did not affect Sertoli cell numbers in any animal group. Both T and DHT increased numbers of spermatogonia and spermatocytes in hpg mice, but DHT has no effect on germ cell numbers in hpg.SCARKO and hpg.ARKO mice. T increased germ cell numbers in hpg.SCARKO and hpg.ARKO mice, but this was associated with stimulation of FSH release. Results show that androgen stimulation of spermatogenesis requires direct androgen action on the Sertoli cells.

Pharmaceutical quality of anthelmintics sold in Kenya.

Nine anthelmintic products purchased in pharmacies and from agricultural merchants in Kenya were tested for pharmaceutical quality. The concentration of active drug was compared with the claim on the label, and the variability of several products was tested between batches and between bottles within the same batch. All the products purchased claimed to contain levamisole but its mean (sd) concentration varied from 0 to 118.0 (13.3) per cent of that claimed. The concentration of levamisole in different batches of the same product ranged from 0 to 85.4 per cent of that claimed. One product consisting in part of mebendazole was found to contain 73.2 (9.4) per cent of the claimed concentration of this active component and two products consisting in part of oxyclozanide were found to contain 106.0 (14.4) and 120.6 (6.1) per cent of the expected concentration of oxyclozanide.

Differentiation of the bovine dominant follicle from the cohort upregulates mRNA expression for new tissue development genes.

This study was designed to identify genes that regulate the transition from FSH- to LH-dependent development in the bovine dominant follicle (DF). Serial analysis of gene expression (SAGE) was used to compare the transcriptome of granulosa cells isolated from the most oestrogenic growing cohort follicle (COH), the newly selected DF and its largest subordinate follicle (SF) which is destined for atresia. Follicle diameter, follicular fluid oestradiol (E) and E:progesterone ratio confirmed follicle identity. Results show that there are 93 transcript species differentially expressed in DF granulosa cells, but only 8 of these encode proteins known to be involved in DF development. Most characterised transcripts upregulated in the DF are from tissue development genes that regulate cell differentiation, proliferation, apoptosis, signalling and tissue remodelling. Semiquantitative real-time PCR analysis confirmed seven genes with upregulated (P< or =0.05) mRNA expression in DF compared with both COH and SF granulosa cells. Thus, the new genes identified by SAGE and real-time PCR, which show enhanced mRNA expression in the DF, may regulate proliferation (cyclin D2; CCND2), prevention of apoptosis or DNA damage (growth arrest and DNA damage-inducible, beta; GADD45B), RNA synthesis (splicing factor, arginine/serine rich 9; SFRS9) and unknown processes associated with enhanced steroidogenesis (ovary-specific acidic protein; DQ004742) in granulosa cells of DF at the onset of LH-dependent development. Further studies are required to show whether the expression of identified genes is dysregulated when abnormalities occur during DF selection or subsequent development.

Maternal Smoking and Fetal Sex Significantly Affect Metabolic Enzyme Expression in the Human Fetal Liver

CONTEXT Pollutants and toxicants passing from the mother to the fetus may damage developing organ systems. The human fetal liver is both a potential target organ and a critical defense against exposure to such xenochemicals. OBJECTIVE The aim of the study was to determine the effects of human fetal toxicant exposure, via maternal smoking, on metabolic enzyme transcripts in the fetal liver. DESIGN AND SETTING We conducted an observational study of mRNA transcripts and proteins in livers from second trimester fetuses at the Universities of Aberdeen and Glasgow. PATIENTS/PARTICIPANTS Liver samples were taken from 55 normal fetuses from women undergoing second trimester elective termination. MAIN OUTCOME MEASURES Housekeeping genes for normalization were identified by GeNorm and NormFinder. Levels of mRNA transcripts encoding 15 metabolic enzymes and three xenobiotic receptors were measured. Expression of representative proteins was shown by Western blotting. RESULTS Eighty-nine percent of measured transcripts were detectable in the human fetal liver. Eight transcripts showed significant sex-specific differences in expression levels (EPHX1, GSTA1, GSTT1, AHR, AS3MT, GLRX2, GGT1, CAR). In male fetuses, maternal smoking was associated with a decrease in expression of three transcripts (GGT1, CYP2R1, CAR) and an increase in eight transcripts (CYP1A1, EPHX1, NQO1, GSTP1, GSTT1, AHR, AS3MT, GLRX2). In the female, CYP3A7 and EPHX1 were increased in smoke-exposed fetuses. CONCLUSIONS The human fetal liver expresses a wide array of metabolic enzymes, with sex differences apparent in 44% of the transcripts measured. Exposure of the fetus to pollutants/toxicants is associated with significantly altered transcript expression, with the more marked response in the male potentially affecting levels of endogenous factors involved in fetal growth.

Sertoli Cells Maintain Leydig Cell Number and Peritubular Myoid Cell Activity in the Adult Mouse Testis

The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR) specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health.

Investigation of the vitamins A and E and beta-carotene content in milk from UK organic and conventional dairy farms.

During a 12-month longitudinal study, bulk-tank milk was collected from organic (n=17) and conventional (n=19) dairy farms in the UK. Milk samples were analysed for vitamin A (retinol), vitamin E (alpha-tocopherol) and beta-carotene content. The farming system type, herd production level and nutritional factors affecting the milk fat vitamin content were investigated by use of mixed model analyses. Conventionally produced milk fat had a higher mean content of vitamin A than organically produced milk fat, although there were no significant differences in the vitamin E or beta-carotene contents between the two types of milk fat. Apart from farming system, other key factors that affected milk fat vitamin content were season, herd yield and concentrate feeding level. Milk vitamin content increased in the summer months and in association with increased concentrate feeding, whilst higher-yielding herds had a lower milk vitamin E and beta-carotene content. Thus, conventional dairy farms in the UK produced milk with a higher vitamin A content, possibly owing to increased vitamin A supplementation in concentrate feeds. However, knowledge of the effects of season, access to fresh grazing or specific silage types and herd production level may also be used by all producers and processors to enhance the vitamin content in milk.