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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. However, the mechanism(s) by which GH facilitated this improvement is yet to be understood. This study aimed to determine these potential mechanism(s) through the use of immunofluorescent localisation of GH receptors (GHRs) on the human oocyte and unbiased computer-based quantification to assess and compare oocyte quality between women of varying ages, with or without GH treatment. This study demonstrates for the first time, the presence of GHRs on the human oocyte. The oocytes retrieved from older women showed significant decrease in the expression of GHRs and amount of functional mitochondria when compared with those from younger patients. More interestingly, when older patients were treated with GH, a significant increase in functional mitochondria was observed in their oocytes. We conclude that GH exerts a direct mode of action, enabling the improvement of oocyte quality observed in our previous study, via the upregulation of its own receptors and enhancement of mitochondrial activity. This result, together with recent observations, provides scientific evidence in support of the use of GH supplementation for the clinical management of poor ovarian response.

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. Conversely, in the older patients this relationship became dysregulated. In the older patients, at the time of preovulatory maturation, the reduced apoptosis reflects the poor mitogenic growth turnover rate of healthy follicles rather than the death rate in an atretic follicle. Restoring an optimum receptor density and down-regulation of receptors may improve oocyte quality and the pregnancy rate in older women.

� Abstract≤O varian folliculogenesis is primarily controlled by the action of gonadotropins namely follicle stimulating hormone (FSH) and luteinizing hormone (L H). Several reports indicated that the process of initial recruitment of primordial follicles to the growing follicles is not gonadotropin-dependent but Bone morphogenetic protein (B M P)-dependent. However, this has not been unequivocally confirmed. T he aim of this study was to investigate the temporo-spacial protein expression of the B M P receptors 1B (B M PR1b), FSH R and L H R in several stages of follicle development. W hile the localization of all receptors was found in granulosa cell membrane of the follicles the temporal expression was varied. B M PR1b was expressed in all follicle stages, FSH R was detected in primary follicles onward and L H R was absent in both primordial and primary follicles but appeared in later stages. Q uantitative analysis based on the intensity of fluorescent signals showed that the expression of B M PR1b, FSH R and L H R significantly (p< 0.001 p< 0.0001 p< 0.0001 respectively) increased with follicular development. W e have concluded that the combination of sensitive immunofluorescence labeling and computerized 3D image analysis proves efficient tools for in situ detection and quantification of the expression of small amount of protein in a complex tissue structure.

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. Many studies have found that in a traditional society with a homogeneous culture, the foremost reason for the different approaches to sex education is related to traditional values, in addition to other factors such as religion and political belief. In order to improve sex education, and consequently, sexual health in a modern multicultural society such as Australia, it becomes imperative to identify the inconsistency in beliefs about sex education among individuals with different cultural backgrounds in the Australian population. In this report, the author highlights similarities and differences in the methods employed by certain cultures of the Australian population. The report considers the different cultural environments of specific societies, the prevalence of sex education in these societies and how culture influences the prevalence. The concluding thoughts reflect on the success of the education programs in Australia, based on the idea that resolving the problems of sex education needs support from a number of bodies within Australian society.

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. Western blot analyses of follicular fluid at day 15 of the cycle showed two strong bands at 42 and 45 kDa and weakly stained bands at 39 and 31 kDa. At day 0, these bands became weaker and the 39 kDa band became undetectable. However, there were no differences in follistatin concentrations between ovaries with and without functional corpus luteum (CL) during the whole luteal phase. In addition, although the ovaries of Booroola ewes normally contain more corpora lutea than those of normal merino ewes, follistatin concentrations in both jugular and ovarian venous blood were similar in Booroola and normal merino ewes. It is concluded that the secretion of follistatin from the ovary is not related to the formation of CL or high ovulation rate of Booroola ewes. The elevation in follistatin concentration in follicular fluid and ovarian blood during late luteal phase may indicate a dual role of follistatin in the luteolysis of existing CL and development of new follicle cohort.

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. The aim of this study was to investigate the role of bone morphogenetic proteins (BMPs) in the regulation of folliculogenesis in mice passively immunised against BMP receptor 1B (BMPRIB) and BMP4. While a stereological study revealed that the numbers of primordial follicles in immunised mice were significantly higher when compared with control animals, treatment with equine chorionic gonadotrophin showed no effect. In parallel, immunofluorescence microscopy revealed the presence of BMPRIB but not FSH receptor in primordial follicles. The number of primary follicles in immunised mice were also significantly increased when compared with control animals. After puberty, the rates of depletion of primordial and primary follicles were increased with age, particularly in treated animals; however, there was no significant difference between the treatment groups of the same age. Based on these results together with our previous reports in sheep and mice, we confirm that the attenuation of BMP signalling system can be an effective approach to sustain the primordial follicle reserve while promoting the development of growing follicles, ovulation and consequently overall female fertility.

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. Any one of these factors, alone or in combination, might lead to an explanation of the discrepancies found in the literature regarding the influence of early changes to the prostate in later life. Therefore, it is important to establish a causal link between the hormonal changes that occur during the fetal/neonatal period and that imprint the gland and the onset of late-life pathology. In order to achieve this goal, several technical challenges need to be overcome to permit the objective assessment of prostate branching morphogenesis. Stereological techniques now allow the quantification of several parameters of branching morphogenesis and the identification of specific early changes that are permanent and irreversible with a late-life outcome. This methodology provides the means to determine the action of a range of genes or hormone/growth factors that have been implicated in prostate development and disease.

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 increased density of FSHR and LHR provide novel evidence of a mechanism for increasing the number of follicles that are recruited during dominant follicle selection. The compounding increase in receptors with increasing follicle size maintained the multiple follicles and reduced the apoptosis, which contributed to a high ovulation rate in BB sheep. In addition, we report a mutation-independent mechanism of down-regulation to reduce receptor density of the leading dominant follicle in sheep. The suppression of receptor density coincides with the cessation of mitogenic growth and steroidogenic differentiation as part of the luteinization of the follicle. The BB mutation-induced attenuation of BMPR1B signaling led to an increased density of the FSHR and LHR and a concurrent reduction in apoptosis to increase the ovulation rate. The role of BMPs in receptor modulation is implicated in the development of multiple ovulations.

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. Since droplets and mitochondria are now known to be attached to intermediate filaments and since these structures are not contractile, it appears to be necessary to invoke the actions of other cytoskeletal elements. Actin microfilaments are involved in cholesterol transport so that it is tempting to propose that the contractile properties of actomyosin are used in this process. It is known that an energy-dependent contractile process involving actin is capable of disrupting intermediate filaments. Since the intermediate filaments appear to act by keeping lipid droplets and mitochondria apart, disruption of the filaments accompanied by a contractile process would be expected to allow these two structures to come together. This would open the way for the transfer of cholesterol to the steroidogenic pathway. This should be regarded as a first step. The events necessary for entry of cholesterol from droplets into the mitochondria remain to be clarified. In addition, the transport process for newly synthesized cholesterol that is not stored in droplets, is still not understood. At least four protein kinase enzymes have been identified in the cytoskeletons of adrenal cells, namely, Ca2+/calmodulin-dependent kinase, protein kinase (Ca2+ and phospholipid-dependent), myosin light chain kinase, and protein kinase A (cyclic AMP-dependent). Tha Ca2+/calmodulin kinase promotes transport of cholesterol to mitochondria and does so under conditions in which phosphorylation of vimentin and myosin light chain occurs. Phosphorylation of vimentin results in disruption of intermediate filaments while phosphorylation of light chain promotes contraction of the actomyosin ring. It now appears that intermediate filaments are cross-linked by actin filaments so that such contraction would be expected to produce significant structural changes in the cytoskeleton and the attached organelles. Although the details of the changes taking place in the organ in vivo are not known, the potential for interaction between droplets and mitochondria as the result of these changes in intermediate filaments and actomyosin, is clear. Protein kinase C is activated by ACTH and cyclic AMP, although this activation does not appear to be directly involved in the regulation of steroid synthesis. Nevertheless, vimentin is a substrate for this enzyme, and changes in the organisation of vimentin filaments and the attached organelles under the influence of protein kinase C have been reported in other cells. Presumably these changes represent part of the response to ACTH because when protein kinase C is activated by phorbol ester, the cytoskeletal changes necessary for rounding up take place but such changes are not accompanied by increased steroid synthesis. Protein kinase A causes rounding of adrenal cells and cytoskeletons. This kinase also causes increased cholesterol transport and, hence, stimulation of steroid synthesis. The enzyme also causes phosphorylation of vimentin but with a different cytoskeletal reorganisation from that seen with the other three kinase enzymes. Clearly phosphorylation plays a major role in these responses. Phosphorylation alters the morphology and the functions of the cytoskeleton and this, in turn, is associated with accelerated cholesterol transport. It is now necessary to define the details of the specific phosphorylation reactions that occur during the response to ACTH, that is, which amino acids are phosphorylated and to what extent by each of the kinase enzymes. Microsc. Res. Tech. 36:463–479, 1997. © 1997 Wiley-Liss, Inc.

The present review demonstrates that the availability of bioactive FSH and LH in PCOS is normal and that granulosa cells of PCO are not apoptotic and instead hyperexpress functional FSH receptors and may possess intact aromatase activity. Consequently, these cells respond excessively to exogenous FSH stimulation and produce high amounts of oestradiol bothin vivoandin vitro. The altered developmental capacity of follicles from PCOin vivois most likely due to the abnormal follicular milieu of PCO and the culminating effects of intrafollicular inhibitors and stimulators. The failure of ovarian oestradiol production and follicular maturation to dominancein vivomay be due to a mechanism that interferes with the function of FSH, such as intraovarian steroids and growth factors. It has previously been shown that EGF and TGFα have inhibitory actions on follicular development, aromatization and LH receptor formation. In contrast, EGF enhances early follicular recruitment and growth. Therefore, it is hypothesized that EGF/TGFα may have a causal relationship in the mechanisms of anovulatory infertility in women with PCOS. Thus, an aberration in the regulation of follicular fluid EGF and/or TGFα may result in reduced numbers of granulosa cells, cessation of follicle selection and ultimately in the creation and maintenance of PCOS. The exact mechanism by which the hyperfunction of EGF/TGFα occurs and the trigger for this hyperactivity in the ovary remain to be determined. An experimental animal model may be required to assist such investigations in the future.

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. Increasing concentrations of EGF and TGF alpha decreased the number of follicles reaching the large antral stage at day 4 of culture. All treatments with EGF, TGF alpha and androstenedione caused significant inhibitions in both follicular growth and oestradiol production, regardless of the dose used (1-20 ng/ml). Moreover, no synergistic effect between androstenedione and either of the two growth factors was observed. These results suggest that EGF, TGF alpha and/or androstenedione may play important roles in the modulation of gonadotrophin-controlled ovarian function. The effects observed in this study suggest that the aberration in the regulation of these growth factors may result in the creation and maintenance of certain ovarian disorders, such as the polycystic ovarian disease.

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. The most intensely staining proteins, using both staining methods, were a double band at 80-87 kDa, and a band at 28.5 kDa, whereas with silver staining, bands at 66.2 kDa and 32.7 kDa were also intensely stained. Electroelution following SDS-PAGE was used to isolate II of these proteins (l16.4, 87.5, 80.9, 66.2, 57.2, 49.7, 46.8, 37.3, 32.7, 28.5 and 15.5 kDa). Amino acid analysis revealed that these proteins were abundant in aspartic and glutamic acid, glycine, serine and leucine, while histidine and phenylalanine were of low abundance. The content of cystine varied widely from 9.4% to 1.4%. The amino termini of the 80.9 kDa, 32.7 kDa, 28.5 kDa and 15.5 kDa proteins were blocked. Immunofluorescence microscopy demonstrated that a polyclonal antiserum to isolated rat fibrous sheath was localized to the principal piece of the rat, rabbit and human spermatozoa, but in the rabbit it also labelled the equatorial region of the head. Western blotting detected all protein bands in isolated fibrous sheath and a similar range of proteins in the spermatozoa of rat and rabbit. Human sperm proteins of 116 kDa and 80 kDa were immunoreactive in common with other species, and there was only weak crossreactivity with the other proteins. These data suggest the presence of common epitopes in the proteins of all three species.

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. In addition, current assessment of oocyte quality after vitrification is commonly based only on the morphological appearance of the oocyte, raising concerns regarding its efficiency. Using both morphological and functional assessments, the present study investigated whether combinations of cryoprotectants at lower individual concentrations result in better cryosurvival rates than single cryoprotectants at higher concentrations. Surplus oocytes from IVF patients were vitrified within 24 h after retrieval using the Cryotop method with several cryoprotectants, either individually or in combination. The morphological and functional quality of the vitrified oocytes was investigated using light microscopy and computer-based quantification of mitochondrial integrity, respectively. Oocyte quality was significantly higher using a combination of cryoprotectants than vitrification with individual cryoprotectants. In addition, the quality of vitrified oocyte varied depending on the cryoprotectants and type of combination used. The results of the present study indicate that observations based purely on the morphological appearance of the oocyte to assess the cryosurvival rate are insufficient and sometimes misleading. The outcome will have a significant implication in the area of human oocyte cryopreservation as an important approach for fertility preservation.

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. Moreover ACTH reorganizes the cytoskeleton and changes the shape of the cell. These observations suggest a mechanism for transport of cholesterol that involves reorganization and contraction of actin microfilaments which may, in turn, cause movement of droplets and mitochondria together through their common attachment to intermediate filaments.

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, 1.5 and 5 years old were collected and processed for the follicle quantification, using computerised stereological methods and statistical analyses. At birth, the total number of primordial follicles in Booroola sheep was significantly lower than in merino sheep. At 1.5 and 5 years, a reversed pattern in favour of Booroola ewes was seen with significantly more primordial follicles than merino. In parallel, the rate of primordial follicle recruitment to developing cohort was substantially lower in Booroola ewes with only 51 and 66% of primordial follicle consumption at 1.5 and 5 years respectively compared to 92 and 97% in merino ewes. On other hand, the mean numbers of developing primary follicles were smaller in Booroola sheep at the time of birth, yet, Booroola ewes possess more primary follicles than merino at 1.5 years. These findings suggest that attenuation of the intraovarian signalling pathway of BMPs may in fact be a successful means of rationalising follicle consumption, preventing unnecessary loss of follicles from the initial primordial follicle pool, hence increasing reproductive longevity and fertility.

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. Patient(s): Forty-two women with normal or polycystic ovaries who attended our facility for the recovery of their immature or mature oocytes or for therapeutic purposes. Intervention(s): Patients underwent intravaginal ultrasound-guided oocyte retrieval or laparoscopic follicular aspiration with or without prior stimulation. Main Outcome Measure(s): Quantitation of EGF receptor in granulosa cells. Result(s): Granulosa cells from polycystic ovaries expressed significantly higher levels of EGF receptor than granulosa cells from normal ovaries. In contrast with patients who were treated with clomiphene citrate, those who were treated with gonadotropins showed low levels of the receptor. However, the levels of the receptor in granulosa cells were not correlated with circulating levels of LH, FSH, progesterone, or E 2 . Immunolabeling of EGF receptor was confined to the cell membrane of granulosa cells. This receptor was fully functional, mediating the ligand-induced inhibition of E 2 production in culture. Conclusion(s): These results provide further evidence supporting a possible role of EGF/transforming growth factor-α in the aberration of ovarian function in polycystic ovary syndrome.

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. The bound filaments appear to be directly adherent to the surface of the droplet and hence take on its curved contour. The binding of the filaments to the droplets was determined by means of tilting. This study provides a new approach to investigate the cytoskeleton and its associated structures with high-resolution three-dimensional images.