Methylation of avpr1a in the cortex of wild prairie voles: effects of CpG position and polymorphism.

DNA methylation can cause stable changes in neuronal gene expression, but we know little about its role in individual differences in the wild. In this study, we focus on the vasopressin 1a receptor (avpr1a), a gene extensively implicated in vertebrate social behaviour, and explore natural variation in DNA methylation, genetic polymorphism and neuronal gene expression among 30 wild prairie voles (Microtus ochrogaster). Examination of CpG density across 8 kb of the locus revealed two distinct CpG islands overlapping promoter and first exon, characterized by few CpG polymorphisms. We used a targeted bisulfite sequencing approach to measure DNA methylation across approximately 3 kb of avpr1a in the retrosplenial cortex, a brain region implicated in male space use and sexual fidelity. We find dramatic variation in methylation across the avrp1a locus, with pronounced diversity near the exon-intron boundary and in a genetically variable putative enhancer within the intron. Among our wild voles, differences in cortical avpr1a expression correlate with DNA methylation in this putative enhancer, but not with the methylation status of the promoter. We also find an unusually high number of polymorphic CpG sites (polyCpGs) in this focal enhancer. One polyCpG within this enhancer (polyCpG 2170) may drive variation in expression either by disrupting transcription factor binding motifs or by changing local DNA methylation and chromatin silencing. Our results contradict some assumptions made within behavioural epigenetics, but are remarkably concordant with genome-wide studies of gene regulation.

Erythrocyte membrane fatty acid composition as a marker of dietary compliance in hyperlipidaemic subjects.

Dietary intervention is the first treatment step in management of hyperlipidaemia, but there are few objective criteria of compliance. Whether intensive dietary intervention would produce a detectable change in erythrocyte membrane fatty acid composition which could be used as a marker of compliance was examined in 31 new hyperlipidaemic patients. Over a 6 month period, body mass index fell from 29.0 to 26.9 kg/m2 (P < 0.001) and total cholesterol by 19% from 8.16 to 6.58 mmol/l (P < 0.001). The energy derived from fat was reduced from 38.5% to 29.6% (P < 0.001), and the ratio of dietary polyunsaturated to saturated (P:S) fatty acids in the diet increased from 0.45 to 0.66 (P < 0.01). Small but significant changes were recorded in several red cell membrane fatty acids, and the P:S ratio increased from 0.91 to 1.13 (P < 0.001). It would appear, therefore, that red cell membrane changes parallel dietary changes and hence are a potential marker for compliance with dietary changes.

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.

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.

Stage-dependent expression of mRNA for cyclic protein 2 during spermatogenesis is modulated by elongate spermatids.

Cyclic protein 2 (CP-2) is a product of the Sertoli cell which is secreted in a cyclical manner according to the stage of the spermatogenic cycle. This study has assessed the influence of the germ cell complement on expression of CP-2 mRNA. Adult rats were treated with 650 mg/kg methoxyacetic acid (MAA) to induce the specific depletion of > 80% of pachytene and later spermatocytes from most tubules, and expression of CP-2 mRNA was then assessed at various times after treatment when particular germ cell types were depleted selectively. CP-2 mRNA was specifically localised to the Sertoli cells of the seminiferous tubules by non-radioactive in situ hybridisation using a digoxigenin-labelled riboprobe. A stage specific variation in CP-2 mRNA levels was observed, with the mRNA being most abundant at stages IV-VII of the spermatogenic cycle. Northern analysis revealed that treatment with MAA led to an apparent increase in the amount of the major 1.7 kb CP-2 transcript when either pachytene spermatocytes or round spermatids were depleted. In contrast, the level of CP-2 mRNA was decreased by more than half at 21 days after MAA treatment. This decrease was confirmed by in situ hybridisation at 21 days after MAA treatment, when CP-2 mRNA expression was found to be decreased or absent from tubules at stages at which CP-2 mRNA is normally expressed (stages IV-VII) when elongate spermatids were depleted selectively from these tubules. These observations lead us to hypothesise that elongate spermatids positively modulate CP-2 mRNA expression in the Sertoli cell.

Bradykinin infusion in chronic cardiac failure and the effects of captopril.

BACKGROUND: Patients with chronic cardiac failure (CCF) have abnormal vascular responses. Bradykinin (BK) is thought to contribute to the vasodilator effects of ACE inhibitors, but the effect of BK itself in patients with CCF has not been examined. METHODS: We studied the responses to infused BK at 10, 30 and 100 pmol min(-1) in patients with CCF (n=10) and controls (n=10). The slope of the dose-response curve was used for comparisons between the groups. Forearm blood flow (FBF) was measured by venous occlusion plethysmography. RESULTS: Following BK, vasodilatation was observed in both groups as the slopes were positive in all, but the difference between the groups was not significant (P=0.77). The study was repeated with the co-administration of 4 micromol min(-1) of N(G)-monomethyl L-arginine (L-NMMA). The vasodilator response to BK was reduced in both groups, and the effect was somewhat greater in the patient group (P=0.23). The vasodilator response to the endothelium-independent vasodilator sodium nitroprusside was slightly less in the patient group (P=0.08). The patients only then underwent repeat infusion of BK before and after a single oral dose of captopril 12.5 mg or placebo. Following captopril, the vasodilator response to BK was unchanged when compared to placebo (difference between slopes, P=0.53). CONCLUSIONS: BK produces dose-dependent vasodilatation in both patients with CCF and controls; there was no difference in the responses, which were antagonised by L-NMMA and therefore in part NO (endothelium)-dependent. The responses were also unchanged after administration of an ACE inhibitor (captopril).

Colocalization of mRNA and protein using in situ hybridization and immunohistochemistry in testicular tissue.

Using testes fixed by perfusion with Bouin's fluid and embedded in paraffin wax, this study has established methods for combining in situ hybridization and immunocytochemistry on the same section to colocalize mRNA and protein for transition protein-1 (TP-1) and sulfated glycoprotein-1 (SGP-1), respectively. It was found that SGP-1 could be detected in tissue sections subsequent to the detection of TP-1 mRNA in situ. The finding that 1) the tissue pretreatments required to permeabilize the section and to allow access to the probe, and 2) the hybridization conditions themselves, had no adverse effect on the detection of antigen, eases the performance of this technique. On this basis, important information could be obtained on the transcriptional and translational activity of spermatogenic cells, if related probes and antibodies are utilized.

Abnormal vascular responses in human chronic cardiac failure are both endothelium dependent and endothelium independent.

OBJECTIVE: To study underlying vascular responses in chronic heart failure in patients without ACE inhibitor treatment, and to compare them with age matched controls. DESIGN: Forearm blood flow was studied using venous occlusion plethysmography in patients with chronic heart failure (n = 12) and matched controls (n = 13), after infusion of L-NMMA (a nitric oxide synthase inhibitor), glyceryl trinitrate (an endothelium independent vasodilator), and serotonin (an endothelium dependent vasodilator). RESULTS: L-NMMA produced significant vasoconstriction in normal subjects (forearm blood flow reduced by 24%), but not in patients (6%; difference between groups p < 0.03). The vasodilator responses to glyceryl trinitrate were impaired in patients (p < 0.02). In normal controls, serotonin produced initial dilatation, followed by vasoconstriction at high doses. In patients, no vasodilator responses were observed, only late vasoconstriction (p < 0.03). CONCLUSIONS: The vascular responses of patients are confirmed as being abnormal. The lack of response to L-NMMA suggests that nitric oxide does not contribute to basal vascular tone in patients with chronic heart failure. The responses to glyceryl trinitrate and to serotonin suggest that there is both smooth muscle and endothelial dysfunction in patients with chronic heart failure.

Impact of diagnosis-related groups on the quality of postoperative care of patients with neck dissections.

Two hundred eighty patients underwent neck dissection over a 10-year period: 138 during the 5-year period before the institution of Diagnosis-Related Group (DRG) reimbursement and 142 during the 5 years after DRG regulations. A comparison of these two groups by site of tumor, stage of disease, histopathology, previous treatment, type of neck dissection, whether neck dissection was carried out alone or in combination with another procedure, presence of preexisting disease, postoperative complications, and mortality revealed no significant differences. A 35% reduction in the length of hospital stay from 16 to 10 days was identified in the post-DRG group with no detrimental effects on patient care. The variables found to have the greatest impact on length of hospital stay were the extent of operation and postoperative complications.

Brucellosis: an unusual cause of fever in Kentucky.

Human infection with brucellosis appears to be very uncommon in Kentucky. Only three cases have been reported to the Kentucky Department of Health, Frankfort, in the last 4 years. It is suspected, however, that brucellosis is severely underdiagnosed and under-reported in the United States. A patient recently diagnosed with acute systemic brucellosis reminds us that this illness may still be seen in the Commonwealth.

Two case study reports of sleep apnoea in patients with paraplegia.

STUDY DESIGN: Case studies of sleep apnoea occurring in two patients with paraplegia. OBJECTIVE: To raise awareness of sleep apnoea in paraplegia. SETTING: Belfast, Northern Ireland. CASE REPORT: We report two patients with paraplegia, one who was having apparent episodes of loss of consciousness and the other daytime somnolence, who were found to have sleep apnoea. The first patient had been medically investigated extensively and a diagnosis of epilepsy was being considered. A joint consultation with the respective partners in each case revealed periods of night-time apnoea and led to sleep study investigations. CONCLUSION: Sleep apnoea is a treatable condition that can occur in patients with paraplegia who are not necessarily obese. Once diagnosed, resolution of symptoms can be rapid and can result in improved quality of life for patients.

Unusual stab injury of the spinal cord.

OBJECTIVE: To report an unusual penetrating stab injury of the spinal cord. DESIGN: Case report of a 13-year-old boy who sustained cervical trauma following an accident while playing. SETTING: Spinal Cord Injuries Unit, Musgrave Park Hospital, Belfast, UK. CASE REPORT: Mechanism of injury was by a spear-like electric fence post entering the neck. Initial neurological examination revealed tetraplegia with C4 sensory level. Magnetic resonance imaging (MRI) of spinal cord demonstrates the penetrating injury. CONCLUSION: No ligamentous instability was demonstrated. In the absence of this, the penetrating injury by a short blade thrown at speed was felt to be responsible for the subsequent injury and resulting outcome at discharge of C4 American Spinal Injury Association (ASIA) grade D tetraplegia.

An audit of the provision of environmental control systems in Northern Ireland, 1992-1997.

OBJECTIVE: To evaluate the provision of environmental control systems (ECS) in Northern Ireland with regard to assessment and prescription, installation and review and propose guidelines for future service delivery. DESIGN: Structured interview, physical examination, Barthel ADL Index, demonstration and assessment of suitability of ECS for patient. SUBJECTS: Prescriptions for ECS from April 1992 to 1997 were identified from centrally held records. Current users were assessed in their own homes. RESULTS: Forty-six out of 49 current users identified were assessed. All were severely disabled (Barthel 0-9); 24% were living alone; 7 (15%) were not utilizing the system; 96% were satisfied with their initial assessment. Prior to prescription 52% had information about ECS and 20% had a practical demonstration; 78% felt that this would have been useful; 52% of users were not under routine clinical review; 41% of ECS had been altered since installation. Repairs had taken longer than 7 days in 11% of cases. In 45 cases the ECS was essential and in 43 it was appropriate to the users' needs and abilities. CONCLUSIONS: ECS are a valuable tool for severely disabled persons and are appropriately prescribed in Northern Ireland. A multidisciplinary team should perform assessment and prescription. All patients should have a practical trial of the equipment to assist in prescriptions. Regular review by the team should be performed to identify changes in need and alter systems appropriately. Users who live alone should represent a priority for repairs.

Follicle-stimulating hormone (FSH) regulates the expression of FSH receptor messenger ribonucleic acid in cultured Sertoli cells and in hypophysectomized rat testis.

FSH acts on Sertoli cells via interaction with a transmembrane receptor (FSHr). Control of expression of the receptor is surely a factor in the regulation of the action of FSH. The regulation of FSHr by FSH and testosterone was studied both in culture and in vivo. Sertoli cells from 18- to 20-day-old male rats were cultured in the presence of 25 ng/ml ovine (o) FSH. At 8 h after addition of FSH, expression of FSHr mRNA decreased significantly. Addition of FSH and actinomycin D to cells did not result in a further decrease in FSHr mRNA levels, suggesting that FSH does not alter turnover of FSHr mRNA. Treatment of cells with 40 ng/ml testosterone did not have any significant effect on the expression of FSHr mRNA. Hypophysectomy of 20-day-old male rats resulted in an increase in expression of FSHr mRNA as compared to that in sham-hypophysectomized animals. This increase was measured at 24 h posthypophysectomy and was maintained at 72 h after surgery. Injection of rats with 0.2 U oFSH at 48 h posthypophysectomy resulted in a reduction in FSHr mRNA when compared to the levels in hypophysectomized rats. Treatment with 2 mg testosterone propionate had no effect on FSHr mRNA levels. The findings confirm that FSH plays an important role in regulating mRNA expression of the FSHr in Sertoli cells in culture and show for the first time that FSHr mRNA is regulated in vivo by FSH in the immature rat testis.

Accumulation of clusterin/sulfated glycoprotein-2 in degenerating pachytene spermatocytes of adult rats treated with methoxyacetic acid.

Clusterin is a ubiquitous glycoprotein that is produced constitutively by Sertoli cells at relatively high amounts. Its association with apoptosis, damage, disease, and repair in nongonadal tissues led us to investigate whether clusterin could be part of a damage-induced response in Sertoli cells brought on by apoptosis of an adjacent cell type. Therefore, the objective of this study was to treat adult rats with methoxyacetic acid (MAA) to selectively destroy pachytene spermatocytes, examine the localization and expression of testicular clusterin, and relate this to the timing of DNA fragmentation, a hallmark of apoptosis. Clusterin protein was localized to the cytoplasm of pachytene spermatocytes at 6 h post-MAA, whereas clusterin mRNA was localized to Sertoli cells. Morphological degeneration of dying cells and DNA fragmentation were not seen until 12 h. Thus, Sertoli cell-derived clusterin had accumulated in the cytoplasm of degenerating spermatocytes early in the apoptotic process. On the basis of these results and the known binding of clusterin to hydrophobic macromolecules, we hypothesize that clusterin is produced by Sertoli cells as a mechanism to "clear" potentially harmful cellular components during the degeneration of germ cells and remodeling of their membranes that occur normally during spermatogenesis.

Cellular localisation of messenger RNAs in rat testis: application of digoxigenin-labelled ribonucleotide probes to embedded tissue.

In an attempt to identify key changes involved in normal spermatogenesis we have developed methods to enable the study of gene expression by the various subpopulations of testicular cells by use of in-situ hybridisation histochemistry. The use of digoxigenin-labelled ribonucleotide and oligonucleotide probes on testicular tissue perfusion-fixed with Bouin's fixative and embedded in paraffin, polystyrene or methacrylate, has been used to accurately localise three transcripts to three different cell types (Sertoli cells, pachytene spermatocytes, and step 7-12 spermatids) within the seminiferous tubule. The ability to produce semi-thin sections of polystyrene- or methacrylate-embedded tissue and successfully to apply digoxigenin-labelled ribonucleotide or oligonucleotide probes resulted in far greater resolution and unequivocal localisation of mRNA in testicular cells than was previously possible by use of thicker paraffin or frozen sections hybridised with 35S-labelled riboprobes. A comparison of the different embedding media versus digoxigenin-labelled oligonucleotide or ribonucleotide probes is made and we demonstrate the relative sensitivities and merits of each combination.

Stage-specific expression of rat transition protein 2 mRNA and possible localization to the chromatoid body of step 7 spermatids by in situ hybridization using a nonradioactive riboprobe.

The present study has used methoxyacetic acid (MAA)-induced depletion of specific germ cell types in the rat and in situ hybridization with nonradioactive riboprobes to determine the stages of the spermatogenic cycle at which there is expression of the mRNA for the basic chromosomal protein transition protein 2 (TP2). On Northern blots, an abundant mRNA was detectable in samples from control adult rats, but the amount of message was markedly reduced when RNA was extracted from the testes of rats treated 14 and 21 days previously with methoxyacetic acid. These testes were depleted specifically of step 7-12 spermatids, suggesting that these cells contain TP2 mRNA. When tissue sections were subjected to in situ hybridization, the TP2 mRNA was localized at the cellular and subcellular levels. Messenger RNA for TP2 was first detectable in spermatids at step 7. In these spermatids, at high magnification, in addition to some positive reaction in the cytoplasm, intense staining was located to a perinuclear structure consistent with localization of mRNA within the chromatoid body. The amount of TP2 mRNA in the cytoplasm increased as remodelling of the early spermatid nucleus progressed and was highest in step 10 and 11 spermatids at stages X and XI. Thereafter, the mRNA decreased until it was undetectable in step 14 spermatids at stage XIV. The localization of TP2 mRNA to the chromatoid body of step 7 spermatids would be consistent with this organelle being a storage site for long-lived mRNAs utilized later in spermiogenesis.

Mouse staufen genes are expressed in germ cells during oogenesis and spermatogenesis.

The Drosophila melanogaster staufen gene encodes an RNA binding protein (Dm Stau) required for the localization and translational repression of mRNAs within the Drosophila oocyte. In mammals translational repression is important for normal spermatogenesis in males and storage of mRNAs in the oocytes of females. In the present study we identified two mouse cDNA expressed sequence tags (ESTs), encoding proteins with significant homology to Dm Stau and used these firstly to screen a mouse kidney cDNA library and secondly to determine whether staufen mRNAs are expressed in the ovaries and testes of mice and rats. Sequence analysis of the cDNAs revealed that they originated from two different genes. Using Northern blots of RNAs from kidneys, ovaries and testes, both cDNAs hybridized to mRNA species of approximately 3 kb in all three tissues. On sections of mouse ovaries, staufen mRNA was localized specifically to oocytes. On sections of mouse testes, staufen mRNA was expressed in spermatocytes found in seminiferous tubules at stages VI-XII of the spermatogenic cycle. In conclusion, we have shown that the mammalian homologues of Dm stau are expressed in germ cells in both male and female mice, consistent with a role for these RNA binding proteins in mammalian gametogenesis.

Localization of mRNAs by in-situ hybridization to the residual body at stages IX-X of the cycle of the rat seminiferous epithelium: fact or artefact?

Several recent articles have reported localization of specific mRNAs in the rat testis to stage IX and X seminiferous tubules using in-situ hybridization. In all cases the expression was located basally in the tubules and appeared as discrete round clusters of grains close to the lamina propria. The localization was interpreted as being in Sertoli cells or leptotene spermatocytes. In this study we demonstrate that this pattern is most probably due to artefactual binding of probes to the residual body (RB). In the present study testicular tissue, perfusion-fixed with Bouin's and embedded in paraffin, was used, as this resulted in excellent morphological preservation such that RBs within tubules at stages VIII-X were clearly distinguishable. RNA content of the RBs was demonstrated at stages VIII-X using methyl green pyronin staining, and could be eliminated by pretreatment with RNAse or trichloroacetic acid. Localization of mRNAs for 11 seminiferous tubule proteins was assessed using 35S-labelled and digoxigenin-labelled riboprobes (activin receptor-II, alpha-inhibin, transferrin, androgen-binding protein (ABP), cyclic protein-2 (CP-2), CREM, sulphated glycoproteins 1 and 2 (SGP-1 and SGP-2), transition protein 2 (TP-2) and cystatin-C), and digoxigenin-labelled oligonucleotide probes (transition protein-1 (TP-1), TP-2 and protamine-1). All of these probes showed localization to the correct cell type(s) within the seminiferous epithelium. In addition, six antisense riboprobes (activin receptor-II, CREM, SGP-2, CP-2, cystatin C and alpha-inhibin) showed hybridization to basally located residual bodies in tubules at stages IX-X on one or more occasions, whereas residual bodies around the edge of the lumen (stage VIII) or in transit through the seminiferous epithelium showed no hybridization; sense probes showed no localization to residual bodies. A common feature of the probes which localized to the basal RBs was that they had been prepared using cDNA cloned into Bluescript SK- vector such that the antisense strand was generated from the T7 polymerase promotor. A cRNA prepared using T7 polymerase and Bluescript vector alone and a GC-rich 27mer oligonucleotide corresponding to the region of the multiple cloning site of Bluescript adjacent to the T7 site both localized uniquely to basal RB. It is concluded that the hybridization seen within RBs is probably a subtle artefact unique to RBs undergoing dissolution following fusion with Sertoli cell lysosomes, and may reflect nonspecific hybridization to GC-rich fragments of RNA.

RNA binding protein Musashi1 is expressed in sertoli cells in the rat testis from fetal life to adulthood.

The Musashi1 (Msi1) gene identified in mouse is a member of a subfamily of RNA binding proteins that are highly conserved across species. Msi1 expression is highly enriched in proliferative cells within the developing central nervous system. Within the testis, proliferation and differentiation of germ cells takes place within the seminiferous epithelium, where these cells are supported physically and functionally by Sertoli cells that do not themselves proliferate following the onset of puberty. RNA binding proteins expressed in testicular germ cells are essential for normal fertility. Preliminary data suggested the mRNA for Msi1 was present in ovary; therefore, we used an Msi1-specific cRNA and monoclonal antibody to investigate whether Msi1 was expressed in the testis. Msi1 mRNA was expressed in rat testis from birth until adulthood; in situ hybridization revealed silver grains within the seminiferous epithelium. Immunohistochemical studies demonstrated that at all ages examined (from Fetal Day 14.5 until adulthood) Msi1 protein was expressed in Sertoli cells. In fetal and adult rat ovaries, Msi1 was detected in granulosa cells and their precursors. In Sertoli cells, protein was detected in both cytoplasmic and nuclear compartments; in adult testes, the immunointensity of the nuclear staining was stage dependent, with highest levels of expression in Sertoli cells at stages I-VI. In rat gonads, the RNA binding protein Msi1 is expressed in both proliferating and nonproliferating Sertoli and granulosa cells.