The effect of ovarian reserve and receptor signalling on granulosa cell apoptosis during human follicle development.

The poor oocyte quality in older women has previously been linked to the depletion of the ovarian reserve of primordial follicles and an increase in granulosal apoptosis. Granulosa cells were collected from 198 follicles and individually analysed by flow cytometry. In the young IVF patients, the level of apoptosis was inversely proportional to the expression of bone morphogenetic protein (BMPR1B) and follicle stimulating hormone (FSH) receptors.

Infertility and ovarian follicle reserve depletion are associated with dysregulation of the FSH and LH receptor density in human antral follicles.

The low take-home baby rate in older women in Australia (5.8%) undergoing IVF (5.8%) is linked to the depletion of the ovarian reserve of primordial follicles.

Dysregulation of granulosal bone morphogenetic protein receptor 1B density is associated with reduced ovarian reserve and the age-related decline in human fertility.

Reproductive ageing is linked to the depletion of ovarian primordial follicles, which causes an irreversible change to ovarian cellular function and the capacity to reproduce. The current study aimed to profile the expression of bone morphogenetic protein receptor, (BMPR1B) in 53 IVF patients exhibiting different degrees of primordial follicle depletion. The granulosa cell receptor density was measured in 403 follicles via flow cytometry.

Flow cytometric analysis of FSHR, BMRR1B, LHR and apoptosis in granulosa cells and ovulation rate in merino sheep.

The aim of the present study was to determine the direct cause of the mutation-induced, increased ovulation rate in Booroola Merino (BB) sheep. Granulosa cells were removed from antral follicles before ovulation and post-ovulation from BB (n=5) and WT (n=12) Merino ewes. Direct immunofluorescence measurement of mature cell surface receptors using flow cytometry demonstrated a significant up-regulation of FSH receptor (FSHR), transforming growth factor beta type 1, bone morphogenetic protein receptor (BMPR1B), and LH receptor (LHR) in BB sheep.

The impact of passive immunisation against BMPRIB and BMP4 on follicle development and ovulation in mice.

The primordial follicle reserve is the corner stone of female fertility and determines the longevity and quality of reproduction. Complete depletion of this reserve will lead to primary infertility, and the key-limiting step of follicle depletion is the transition from primordial to primary follicles. It has been reported that this process is gonadotrophin-independent, but other conflicting reports are indicated otherwise and this discrepancy needs to be unequivocally clarified.

A direct action for GH in improvement of oocyte quality in poor-responder patients.

Declining female fecundity at later age and the increasing tendency for women to delay childbirth have lead to a drastic rise in the number of women seeking assisted reproductive technology. Many women fail to respond adequately to standard ovarian stimulation regimens, raising a significant therapeutic challenge. Recently, we have demonstrated that the administration of GH, as an adjunct to ovarian stimulation, has improved the clinical outcomes by enhancing the oocyte quality.

Multiculturalism and inconsistency in the perception of sex education in Australian society.

A group of individuals who share common beliefs form a culture in which they communicate their values and attributes about certain aspects of society. Sex education remains one of the early teachings that humans experience irrespective of the race or level of development of a given society. However, different cultures perceive sex education differently due to differences in attitudes and beliefs, leading to significant diversity in the management of sex education among different societies across the globe.

Optimising vitrification of human oocytes using multiple cryoprotectants and morphological and functional assessment.

Oocyte vitrification is a clinical practice that allows preservation of fertility potential in women. Vitrification involves quick cooling using high concentrations of cryoprotectants to minimise freezing injuries. However, high concentrations of cryoprotectants have detrimental effects on oocyte quality and eventually the offspring.

Hypo-osmotic swelling test identifies individual spermatozoa with minimal DNA fragmentation.

One concern during intracytoplasmic sperm injection (ICSI) is that selected spermatozoa may have increased levels of DNA damage; however, the available testing for this is largely destructive in nature and therefore unsuitable as a tool for sperm selection. One alternative selection process that has previously achieved pregnancies is the hypo-osmotic swelling test (HOST). This study reports that low HOST values of neat semen samples were significantly (P<0.

Ovarian follicle development in Booroola sheep exhibiting impaired bone morphogenetic protein signalling pathway.

The role of bone morphogenetic proteins (BMPs) in the regulation of ovarian function has been extensively investigated but the mechanism of regulation is not well understood. The aim of this study was to investigate the effect of mutation in the BMP receptor in Booroola sheep on the number of primordial follicles and rate of follicle recruitment in comparison with that in normal merino sheep in vivo. Whole sheep ovaries at the time of birth, 1.

Changes in ovarian follistatin levels during the oestrous cycle in sheep may serve as an intraovarian regulator.

The expression and concentration of follistatin and activin change during oestrous cycle suggesting their involvement in the regulation of follicular development. The aim of this study was to determine the level, source and potential role of follistatin in the sheep ovary. Follistatin in ovarian venous blood, measured by radioimmunoassay, remained at its low level from follicular phase (day -1 and 0) to mid-luteal phase (days 11-13) phase but were significantly elevated during the late luteal phase (days 14 and 15) when corpora lutea underwent regression.

Early prostate development and its association with late-life prostate disease.

The development of the prostate is an emerging priority area for prostate biologists. Early changes in prostate development permanently alter prostate morphology and function and an understanding of the permanent nature of early events that may influence the onset of late-life disease is vital. Two of the inherent problems involve associating exposure in early life with outcome in late life or maturity and accounting for the influence of genetic, environmental, dietary or metabolic factors during the intervening period.

Computer-based detection of neonatal changes to branching morphogenesis reveals different mechanisms of and predicts prostate enlargement in mice haplo-insufficient for bone morphogenetic protein 4.

Early changes to branching morphogenesis of the prostate are believed to lead to enlargement of the gland in adult life. However, it has not been possible to demonstrate directly that alterations to branching during the developmental period have a permanent effect on adult prostate size. In order to examine branching morphogenesis in a quantitative manner in neonatal mice, a combination of imaging and computational technology was used to detect and quantify branching using bone morphogenetic protein 4 haplo-insufficient mice that develop enlarged prostate glands in adulthood.

Isolation and characterization of rat sperm tail outer dense fibres and comparison with rabbit and human spermatozoa using a polyclonal antiserum.

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 mechanism of action of epidermal growth factor and transforming growth factor alpha on aromatase activity in granulosa cells from polycystic ovaries.

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 (P< 0.001) greater amounts of oestradiol-17beta than that of granulosa cells from normal ovaries.

Hyperexpression of epidermal growth factor receptors in granulosa cells from women with polycystic ovary syndrome.

Objective: To compare the localization and quantitation of epidermal growth factor (EGF) receptor in granulosa cells from women with normal and polycystic ovaries.

Design: Controlled, comparative study.

Setting: Academic research laboratory.

Roles of microfilaments and intermediate filaments in adrenal steroidogenesis.

The problem for the steroidogenic cell if it is to accelerate steroid synthesis in response to trophic stimulation, consists in moving cholesterol from the sites of synthesis and storage to mitochondria at an accelerated rate. The most intensely studied situation is that in which the sterol is stored as ester in lipid droplets. Cholesterol ester must be de-esterified and transported to mitochondria where steroid synthesis begins.

Functional integrity of 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.

Effects of epidermal growth factor, transforming growth factor alpha and androstenedione on follicular growth and aromatization in culture.

The effects of epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) and androstenedione on follicular growth and oestradiol production were studied in cultured mouse preantral follicles. Cultured follicles in the control group showed growth and steroidogenic capacities up to the antral follicle stage at day 4 of culture. However, many of these follicles failed to resume their growth during the culture.

Adhesion of intermediate filaments and lipid droplets in adrenal cells studied by field emission scanning electron microscopy.

High-resolution field emission scanning electron microscopy was used to study the organisation of intermediate filaments around lipid droplets and their binding to these droplets, in primary culture of bovine adrenal cells. Whole-mount preparations of intermediate filaments and bound lipid droplets were prepared from cells grown on Formvar-coated grids and processed by freeze-drying. Intermediate filaments were seen as an interconnected network enveloping the entire droplet.

Isolation and partial characterization of rat sperm tail fibrous sheath proteins and comparison with rabbit and human spermatozoa using a polyclonal antiserum.

Rat sperm tail fibrous sheath was isolated using mechanical and chemical dissection methods from spermatozoa collected from the cauda epididymis. The procedures used to isolate the fibrous sheath were monitored by phase-contrast microscopy and purity was verified by electron microscopy. SDS-PAGE of isolated total fibrous sheath revealed at least 17 bands when stained with Coomassie brilliant blue and 20 bands with silver stain.

Calcium/calmodulin induces phosphorylation of vimentin and myosin light chain and cell rounding in cultured adrenal cells.

In previous reports on adrenal cells we have shown that calcium/calmodulin regulates cholesterol transport to mitochondria and induces phosphorylation of cytoskeleton homogenates. In this study, we have used bovine fasciculata cells permeabilized in situ to identify the phosphorylated proteins and to investigate the manner in which the cytoskeleton components may act together in any subsequent reorganization of the cell. The main cytoskeletal proteins namely vimentin, tubulin, actin, and the associated protein myosin light chain were identified on polyacrylamide gel electrophoresis by their molecular weights and by Western blotting using affinity-purified monoclonal antibodies.

Binding of lipid droplets and mitochondria to intermediate filaments in rat Leydig cells.

We have examined the distribution of lipid droplets and mitochondria in relation to the cytoskeletons of Leydig cells in primary culture by using light and electron microscopy on living, intact and detergent-extracted cells. After mild extraction with Triton X-100 lipid droplets and mitochondria retained their original distribution within the cell. Double immunofluorescent microscopy showed that both structures co-localise with intermediate filaments.

Indirect immunofluorescence modified to display two antigens with one light filter.

We present a modification of double indirect immunofluorescence in which we used four antibodies raised in three species to visualize two different antigens. The procedure, which relies on dual recognition of a secondary antibody, requires that one primary antibody and one of the secondary antibodies be raised in the same species. As the two secondary antibodies are conjugated to two different fluorochromes, both of the antigens studied are visualized with one light filter while only one antigen is displayed with another filter.

The role of the cytoskeleton in the regulation of steroidogenesis.

The slow step in steroid synthesis involves the transport of cholesterol from lipid droplets in the cytoplasm to the first enzyme in the pathway-the cytochrome P450 that converts cholesterol to pregnenolone (P450scc) which is located in the inner mitochondrial membrane. ACTH stimulates this intracellular transport of cholesterol in adrenal cells (Y-1 mouse adrenal tumour cells and cultured bovine fasciculata cells) and this effect of the trophic hormone is inhibited by cytochalasins, by anti-actin antibodies and DNase I suggesting that the response to ACTH requires a pool of monomeric (G-) actin that can be polymerized to F-actin. Recent studies have shown that lipid droplets and mitochondria of adrenal cells are both attached to intermediate filaments.