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The rate of steroid synthesis is regulated by the rate of transport of cholesterol from lipid droplets to mitochondria. We have previously demonstrated that lipid droplets in adrenal cells are tightly attached to intermediate filaments. Here we now show that mitochondria colocalize with intermediate filaments in modified double indirect immunofluorescence and by electron microscopy of extracted adrenal cells. Direct contact between mitochondria and intermediate filaments was established by examination of stereo pairs of electron micrographs from extracted cells. The attachment of both droplets and mitochondria to intermediate filaments suggests possible mechanisms for this form of intracellular transport of cholesterol to mitochondria and hence for the regulation of steroid synthesis.

The development and growth of the prostate gland requires branching morphogenesis, a process that continues during the neonatal period. Estrogen is involved in prostate development but its temporospatial effects during early development are not understood. Using new methods of confocal microscopy and volume-rendering image analysis, the aim was to detect and quantify the temporospatial changes in prostate branching morphogenesis in neonatal estrogen-deficient ArKO mice. In wild type animals, the lateral and medial ducts of anterior prostatic lobes reveal developmental asymmetry with significant increases in branching morphogenesis of the lateral duct, which is retained during day 0-3. In ventral lobes, similar asymmetrical growth is observed but increases in branching occur randomly in lateral or medial ducts. At day14, ArKO mice exhibit prostatic hypertrophy and significant increases in the stromal, epithelial and lumenal volume. To determine whether estrogen deficiency has specific immediate effects on neonatal prostate branching morphogenesis, prostatic lobes were analysed at days 1 and 3. At day 1, anterior lobe from knockout mice exhibits significant increase in the duct volume only. At day 3, all events of branching morphogenesis are significantly accelerated in both knockout and heterozygous mice. In the ventral lobe, the effect of estrogen deficiency results in significant increases in some branching parameters in knockout animals at day 3 only. To determine the spatial distribution of these alterations within prostatic lobes, branching events were assessed in individual ducts. In the anterior lobe, all parameters are significantly increased in both lateral and medial ducts in knockout animals with significantly pronounced increases in the medial ducts, resulting in less asymmetry. In the ventral lobe, however, no significant alteration is found in either duct. In summary, estrogen deficiency in ArKO mice leads to significant neonatal age-related lobeand ductspecific alterations; being more detrimental in anterior than in ventral lobes and more pronounced in medial than in lateral ducts. We conclude that estrogen is a critical regulator of prostate gland development, exerting significant early effects on gland growth. Therefore, the detection and quantification of aberrant branching morphogenesis within the first 4 days of neonatal life predicts prostate pathology that is known to occur in adulthood.

We investigated aromatization and the mechanism of action of epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) on oestradiol biosynthesis in freshly prepared granulosa cells from polycystic ovaries. Freshly prepared granulosa cells from polycystic ovaries incubated for only 3 h under basal conditions secreted significantly (P0.001) greater amounts of oestradiol-17beta than that of granulosa cells from normal ovaries. 8-Bromo-cyclic adenosine monophosphate (8-Br-cAMP), but not follicle stimulating hormone (FSH) or luteinizing hormone (LH), further enhanced this activity. Both EGF and TGFalpha inhibited gonadotrophinor 8-Br-cAMP-stimulated, but not basal, oestradiol production. LH receptor (LHR) binding, estimated by immunolabelling the bound LH, was significantly (P0.001) reduced in granulosa cells from polycystic ovaries when compared with cells from normal ovaries. EGF or TGFalpha significantly reduced the binding in cultured cells from all patient groups (P0.05). More interestingly, a further increase of the inhibitory effect was seen in granulosa cells from polycystic ovaries (P0.001). In conclusion, granulosa cells from polycystic ovaries contain high levels of basal aromatase activity in vitro, which is probably inherited from the in-vivo condition. EGF and TGFalpha suppress oestradiol synthesis at a step beyond the production of cAMP and also LHR binding with more effect in granulosa cells from polycystic ovaries.

OBJECTIVE The polycystic ovarian syndrome is frequently associated with human infertility and is a partially characterized syndrome of unknown aetiology. The aim of this study was to describe the functional integrity of granulosa cells from polycystic ovaries. PATIENTS Follicular aspirates were collected from polycystic ovaries of ovulatory (n = 24) and anovulatory (n = 7) patients. Follicular aspirates were also collected from normal ovaries of untreated (n = 24) and superovulated (n = 10) subjects. All patients were enrolled for the recovery of their oocytes for in vitro maturation and fertilization. MEASUREMENTS FSH receptors and apoptosis were measured in the granulosa cells of the different patients. FSH-stimulated oestradiol and LH-stimulated progesterone production by granulosa cells of the different patients were also measured. RESULTS The binding of 125I-labelled human recombinant FSH to granulosa cells from anovulatory subjects with polycystic ovaries was significantly higher than that found in granulosa cells from normal (180%) and superovulated (163%) ovaries. However, the ligand binding to granulosa cells from ovulatory subjects with polycystic ovaries was not significantly higher than that found in normal granulosa cells. Also, granulosa cells obtained from anovulatory subjects with polycystic ovaries cultured with FSH produced more oestradiol than normal granulosa cells but oestradiol production was similar to that of granulosa cells from superovulated ovaries (mean ± SEM, 244.94 ± 22.02, 24.23 ± 2.92, 211.87 ± 50.39 nmol/l/24 h, respectively). Flow cytometric analysis showed that the proportions of viable and apoptotic granulosa cells (mean ± SDM, 70 ± 5 and 7 ± 1%, respectively) were similar in normal subjects and in those with polycystic ovaries. CONCLUSION We conclude that most of the granulosa cells of polycystic ovaries are healthy and non-apoptotic, expressing high levels of FSH receptors and highly responsive to this hormone in culture. These data provide direct evidence that most of the follicles of polycystic ovaries are not atretic.

Rat outer dense fibres were isolated from cauda epididymal spermatozoa using mechanical and chemical dissection methods. Sperm tail isolation procedures were monitored by phase-contrast microscopy and the purity of the outer dense fibres was verified by electron microscopy. SDS-PAGE of isolated outer dense fibres revealed at least nine Coomassie brilliant blue stained bands, and 12 silver staining bands. The most abundant proteins were a large band between 26.5 and 32.5 kDa, and 84 kDa, 21.5 kDa and 15.5 kDa bands. The amino acid composition of the total rat outer dense fibres and seven isolated proteins showed similar compositions, being abundant in aspartic and glutamic acid, serine, glycine and leucine. However, the content of cysteine and proline was highly variable among the isolated proteins. Immunofluorescence microscopy demonstrated that a polyclonal antiserum to isolated rat outer dense fibres showed positive staining localized to the mid-piece of rat and rabbit spermatozoa. However, there was crossreactivity in the principal piece as well as the mid-piece of the human spermatozoa. The antiserum also showed crossreactivity in the perforatorium of rat sperm heads and the acrosome and equatorial segment of rabbit sperm heads. These data indicate that it is technically possible to isolate proteins from the outer dense fibres that will enable further studies of the amino acid sequences of sperm tail proteins.