Advancing Methods of Assessing Bone Quality to Expand Screening for Osteoporosis.

CONTEXT: Dual-energy x-ray absorptiometry (DXA) limits osteoporosis screening because of machine size, technical requirements for operation, and exposure to ionizing radiation. OBJECTIVE: To establish data ranges from calcaneus ultrasonography (US) that correspond to bone mineral density (BMD) stratification identified by DXA and to determine whether vitamin D concentration adds to US bone health assessment. METHODS: Patients scheduled for DXA at the Robert C. Byrd Clinic, a rural primary care facility in Lewisburg, West Virginia, were recruited from June 2015 to June 2016. Ultrasonography was used to scan the left and right calcaneus of the patients, and blood was collected from a finger prick for vitamin D analysis. Information was collected regarding Fracture Risk Assessment tool parameters, menstrual history, and drug and supplement use. The correlations within and between DXA and US measurements were calculated, as well as the correlations between DXA and US measurements and vitamin D levels. Predictive performance of US readings on bone health determined by DXA scan was assessed with area under the curve analysis using receiver operator characteristic curves. RESULTS: Ninety-nine participants were included. Ultrasonography readings of either the left or right foot were predictive of good vs poor bone quality. No differences were found between US scans of the left foot vs the right foot. Area under the curve values for US BMD T scores for the left and right foot were 0.69 and 0.68, respectively. There was no correlation between DXA- and US-assessed BMD and vitamin D concentrations. Negative correlations were observed between the DXA BMD T scores and vitamin D concentration of the spine and right hip; negative correlations were also observed in the Z score from the spine in the subset of participants who reported not taking vitamin D supplements. CONCLUSION: Ultrasonography of the calcaneus offers a low-cost, efficient means to screen bone health. The affordability and mobility of a US machine enables its use as a screening method that may be applicable to large numbers of people. This study established a T score greater than -1.05 as an indicator of good bone quality and a T score less than -1.05 as an indicator of poor bone quality when using US for BMD screening.

Immunohistochemical techniques to identify and localize proteins of interest in paraffin embedded tissue sections.

Proteins carry out cellular functions. Identifying proteins within tissues, which are characteristically comprised of various cell types, is critical to understanding how the tissue functions. Being able to assess protein expression in tissues is also essential to gaining insight into how tissues change under different physiological conditions, in pathological states, in response to treatments, etc. Immunohistochemistry exploits antibody-antigen associations to identify specific proteins within tissues. This is a very powerful technique as it allows for studying tissues intact, preserving cellular relationships, and tissue structure. Here, we discuss the process of using an antibody specific for PPARγ to identify ovarian cells that express this transcription factor, and how its expression changes during the ovarian cycle.

Initiation of the expression of peroxisome proliferator-activated receptor gamma (PPAR gamma) in the rat ovary and the role of FSH.

PPARgamma is highly expressed in granulosa cells by 23 days post-partum (pp) and is down-regulated in response to the LH surge. We tested the hypothesis that high levels of FSH during the neonatal period trigger the expression of PPARgamma. To determine when PPARgamma expression is initiated, ovaries were collected from neonatal rats. Messenger RNA for PPARgamma was undetectable on day 1, low from days 5-14, and increased by day 19 pp (p < 0.05). PPARgamma was detected in select granulosa cells in primary/early secondary follicles. Messenger RNA for the FSH receptor was detected as early as day 1 and remained steady throughout day 19 pp. The FSH receptor was detected by immunoblot analysis in ovaries collected 1, 2, and 5-9 days pp. In a subsequent experiment, neonatal rats were treated with acyline (GnRH antagonist) which significantly reduced FSH (p < 0.05) but not levels of mRNA for PPARgamma. The role of FSH in the induction of PPARgamma expression was further assessed in ovarian tissue from FORKO mice. Both mRNA and protein for PPARgamma were identified in ovarian tissue from FORKO mice. In summary, the FSH/FSH receptor system is present in granulosa cells prior to the onset of expression of PPARgamma. Reducing FSH during the neonatal period, or the ability to respond to FSH, did not decrease expression of mRNA for PPARgamma. These data indicate that FSH is not a primary factor initiating the expression of PPARgamma and that other agents play a role in activating its expression in the ovary.

Polarized ovaries of the long-tongued bat, Glossophaga soricina: a novel model for studying ovarian development, folliculogenesis, and ovulation.

Glossophaga soricina is a spontaneously ovulating, monovular, polyestrous bat with a simplex uterus, exhibiting true menstruation. Studies conducted on reproductively active, captive-maintained animals established that G. soricina also has polarized ovaries, with the ovarian surface epithelium (OSE) restricted to the medial side of the ovary, and primordial follicles limited to an immediately adjacent zone. Follicles selected for further development are recruited from the medullary side of this zone, and ovulation is restricted to the portion of the ovary covered by the OSE. To further develop G. soricina as a model for studying ovarian development and physiology, ovaries were collected from fetal, neonatal, and adult females and processed for morphological and immunohistochemical analyses. The latter included staining for factor VIII-related antigen (von Willebrand factor) to assess regional differences in ovarian vascularity. The ovarian structure in fetal and neonatal animals was very similar to that in other species that do not have polarized ovaries at comparable stages of development. This indicated that polarization of the ovary does not occur during fetal development, but rather sometime between the neonatal period and adulthood. Vascular elements were abundant in areas of the ovary surrounding early growing follicles, but sparse in the zone of the ovary containing primordial follicles. The polarized nature of the ovaries in G. soricina suggests that this species might be used as a model to investigate the formation, long-term maintenance, and activation of primordial follicles, and the role of the OSE in ovulation and folliculogenesis.

Potential role for peroxisome proliferator-activated receptor gamma in regulating luteal lifespan in the rat.

Peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to stimulate progesterone production by bovine luteal cells. We previously reported higher expression of PPARgamma in old compared with new luteal tissue in the rat. The following studies were conducted to determine the role of PPARgamma in rat corpora lutea (CL) and test the hypothesis that PPARgamma plays a role in the metabolism of progesterone and/or luteal lifespan. Ovaries were removed from naturally cycling rats throughout the estrous cycle, and pseudopregnant rats. mRNA for PPARgamma and P450 side-chain cleavage (SCC) was localized in luteal tissue by in situ hybridization, and protein corresponding to PPARgamma and macrophages identified by immunohistochemistry. Luteal tissue was cultured with agonists (ciglitazone, prostaglandin J2) or an antagonist (GW-9662) of PPARgamma. Progesterone was measured in media by RIA and levels of mRNA for 20alpha-hydroxysteriod dehydrogenase (HSD) and bcl-2 were measured in luteal tissue after culture by RT-PCR. An inverse relationship existed between the expression of mRNA for SCC and PPARgamma. There was no effect of PPARgamma agonists or the antagonist on luteal progesterone production in vitro, or levels of mRNA for 20alpha-HSD. PPARgamma protein was localized to the nuclei of luteal cells and did not correspond with the presence of macrophages. In new CL, ciglitazone decreased mRNA for bcl-2 on proestrus, estrus, and metestrus. Interestingly, GW-9662 also decreased mRNA for bcl-2 on proestrus and diestrus in old and new CL, and on metestrus in new CL. These data indicate that PPARgamma is not a major player in luteal progesterone production or metabolism but may be involved in regulating luteal lifespan.

Effects of luteinizing hormone on peroxisome proliferator-activated receptor gamma in the rat ovary before and after the gonadotropin surge.

We have shown previously that mRNA for peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed in granulosa cells and downregulated by the luteinizing hormone (LH) surge. The current studies were undertaken to test the hypothesis that LH stimulates a decrease in the expression of PPARgamma, as well as its activity, in granulosa cells. Ovaries were collected from immature rats 0 and 48 h after they received pregnant mares' serum gonadotropin (PMSG), and 4 and 24 h after administration of human chorionic gonadotropin (hCG), and used for protein isolation or processed for immunolocalization of PPARgamma. The amount of phosphorylated PPARgamma was measured by immunoblot analysis to determine how LH affects the phosphorylation status, and therefore the activity, of PPARgamma. Granulosa cells were also collected from immature rats 48 h after PMSG. Cells were cultured with LH in the absence and presence of H89 and cycloheximide to investigate the role of PKA and protein synthesis in the LH-mediated decline in mRNA for PPARgamma respectively. Protein corresponding to PPARgamma was localized to nuclei of granulosa cells 0 and 48 h after PMSG. Expression was greatly reduced by 4 h after hCG, with expression in mural granulosa cells lost before that in cumulus cells. The amount of phosphorylated PPARgamma did not change during the periovulatory period. Blocking PKA activity had no effect on levels of mRNA for PPARgamma. However, levels of mRNA for PPARgamma were significantly increased in cells treated with cycloheximide (P < 0.05, ANOVA followed by Tukey's HSD). These data suggest that PPARgamma is tightly regulated in the ovary and that its expression is the primary mechanism by which LH influences the activity of PPARgamma. In addition, protein synthesis may be involved in modulating levels of PPARgamma in granulosa cells.

Peroxisome proliferator-activated receptors (PPARs) and ovarian function--implications for regulating steroidogenesis, differentiation, and tissue remodeling.

The peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors involved in varied and diverse processes such as steroidogenesis, angiogenesis, tissue remodeling, cell cycle, apoptosis, and lipid metabolism. These processes are critical for normal ovarian function, and all three PPAR family members--alpha, delta, and gamma, are expressed in the ovary. Most notably, the expression of PPARgamma is limited primarily to granulosa cells in developing follicles, and is regulated by luteinizing hormone (LH). Although much has been learned about the PPARs since their initial discovery, very little is known regarding their function in ovarian tissue. This review highlights what is known about the roles of PPARs in ovarian cells, and discusses potential mechanisms by which PPARs could influence ovarian function. Because PPARs are activated by drugs currently in clinical use (fibrates and thiazolidinediones), it is important to understand their role in the ovary, and how manipulation of their activity may impact ovarian physiology as well as ovarian pathology.

Expression of messenger RNA for ADAMTS subtypes changes in the periovulatory follicle after the gonadotropin surge and during luteal development and regression in cattle.

Extensive remodeling of the extracellular matrix occurs in the ovary during the periovulatory period. Matrix metalloproteinases and their endogenous inhibitors, tissue inhibitors of metalloproteinases, are believed to play integral roles in this highly regulated series of cellular events, but their specific roles remain unclear. Recent cloning studies have identified a novel family of metalloproteinases, the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family. The regulated expression of distinct ADAMTS subtypes has been shown to be required for tissue morphogenesis during embryonic development and for maintaining the integrity of tissues in the adult. In the present studies, we have determined that multiple ADAMTS subtypes are present in the bovine ovary using a reverse transcription-polymerase chain reaction strategy. In particular, ADAMTS-1, -2, -3, -4, -5 (also known as ADAMTS-11), -7, -8, and -9, but not ADAMTS-6, -10, or -12, mRNA transcripts were detected in granulosa cells of nonatretic ovarian follicles and corpora lutea. The levels of mRNA for these ovarian ADAMTS were up- or down-regulated or remained unchanged in the granulosa and/or theca cells of the dominant follicle following the preovulatory surge of gonadotropins, depending on the subtype and/or the cell compartment, and in the corpus luteum during the luteal phase of the estrous cycle. The complex expression patterns observed for the distinct ADAMTS subtypes in the granulosa and theca cells of the periovulatory follicle and in the luteal tissues of the bovine ovary suggest that these novel proteases mediate, at least in part, the remodeling events underlying folliculogenesis and ovulation and the formation, maintenance, and regression of the corpus luteum.

Inverse relationship between the expression of messenger ribonucleic acid for peroxisome proliferator-activated receptor gamma and P450 side chain cleavage in the rat ovary.

Messenger RNA for peroxisome proliferator-activated receptor gamma (PPARgamma) has been found in granulosa cells, and its expression decreases after the LH surge. We determined which developmental stage of ovarian follicle expresses mRNA for PPARgamma and evaluated the impact of PPARgamma agonists on steroidogenesis. Ovaries were collected from immature eCG/hCG-treated rats at 0 (no eCG), 24, and 48 h post-eCG and 4 and 24 h post-hCG. Ovarian tissue was serially sectioned and processed for in situ hybridization to localize mRNA corresponding to PPARgamma, aromatase, and the LH receptor, and P450 side chain cleavage (P450SCC) and to determine whether apoptotic cells were present. During follicular development, there was no correlation between the expression of mRNAs for PPARgamma and aromatase or the presence of apoptotic cells, but a general inverse correlation was observed between the expression of PPARgamma mRNA and LH receptor mRNA. At 4 h post-hCG, follicles expressing P450SCC mRNA had lost expression of PPARgamma mRNA. This inverse pattern of expression between PPARgamma and P450SCC mRNAs was also observed 24 h post-hCG, with developing luteal tissue expressing high levels of P450SCC mRNA but little or no PPARgamma mRNA. To determine the impact of PPARgamma on steroidogenesis, granulosa cells were collected from ovaries 24 h post-eCG and cultured alone, with FSH alone, or with FSH in combination with the PPARgamma agonists ciglitazone or 15-deoxy-delta 12,14-prostaglandin J2 (PGJ2). Treatment of granulosa cells with PGJ2 stimulated basal progesterone secretion, whereas ciglitazone or PGJ2 had no significant effect on FSH-stimulated steroid production. These findings suggest that 1) PPARgamma may regulate genes involved with follicular differentiation and 2) the decline in PPARgamma in response to LH is important for ovulation and/or luteinization.

Localization and expression of tissue inhibitor of metalloproteinase-4 in the immature gonadotropin-stimulated and adult rat ovary.

The tissue inhibitors of metalloproteinases (TIMPs) are important regulators of the matrix metalloproteinases (MMPs), proteolytic enzymes essential for controlling the coordinated tissue remodeling that takes place in the ovary. In the present study, we characterized the ovarian expression pattern of TIMP-4. The localization of TIMP-4 mRNA was determined by in situ hybridization in adult cycling rats. TIMP-4 mRNA on the day of estrus was expressed in a punctate pattern in stroma and in corpora lutea (CL) from previous cycles but not in newly formed CL or follicles. At metestrus, TIMP-4 mRNA was present in certain CL from the current and previous cycles and continued to exhibit a punctate pattern of expression in the stroma. By diestrus, TIMP-4 mRNA was detected in the thecal layer surrounding follicles, and a relatively high level of expression was observed in a punctate pattern within new and previous CL and in the stroma. TIMP-4 mRNA was also observed in the thecal layer at proestrus, but the punctate pattern within CL and stroma was absent. To correlate the changes in cellular localization with changes in overall TIMP-4 levels, ovarian mRNA and protein levels were examined in adult cycling rats and in gonadotropin-stimulated immature rats. In cycling rats, there was no change in mRNA or protein levels across the cycle, although there was a trend towards higher levels during estrus (P = 0.08). In gonadotropin-treated rats, there was an increase in TIMP-4 mRNA 48 h after eCG administration with a corresponding doubling of TIMP-4 protein. Although TIMP-4 mRNA and protein tended to decline after hCG treatment, this trend was not significant (P = 0.08). These findings indicate that TIMP-4 could play an important role in regulating MMPs in a localized manner in follicles and CL throughout the cycle.

Localization and expression of messenger RNAs for the peroxisome proliferator-activated receptors in ovarian tissue from naturally cycling and pseudopregnant rats.

Structural and functional development of the corpus luteum (CL) involves tissue remodeling, angiogenesis, lipid metabolism, and steroid production. The peroxisome proliferator-activated receptors (PPARs) have been shown to play a role in these as well as in a multitude of other cellular processes. To examine the expression of mRNA corresponding to the PPAR family members (alpha, delta, and gamma) in luteal tissue, ovaries were collected from gonadotropin-treated, immature rats on Days 1, 4, 8, and 14 of pseudopregnancy and from adult, cycling animals on each day of the estrous cycle. Ovaries were processed for in situ hybridization or RNA isolation for analysis by RNase protection assay. The expression of PPARgamma mRNA was abundant in granulosa cells of developing follicles during both pseudopregnancy and the estrous cycle and was low to undetectable in CL from pseudopregnant rats. However, luteal tissue in cycling animals, especially CL remaining from previous cycles, had high levels of PPARgamma mRNA. The PPARalpha mRNA was localized mainly in the theca and stroma, and PPARdelta mRNA was expressed throughout the ovary. Levels of mRNA for PPARgamma decreased between Days 1 and 4 of pseudopregnancy, and PPARalpha mRNA levels were lower on the day of estrus compared to pro- and metestrus (P < 0.05). The PPARdelta mRNA levels remained steady throughout the estrous cycle and pseudopregnancy. These data illustrate a difference in the luteal expression of mRNA for PPARgamma between the adult, cycling rat and the immature, gonadotropin-treated rat. This differential pattern of expression may be related to the difference in timing of the preovulatory prolactin surge, because the gonadotropin-primed animals would not experience a prolactin surge coincident with the LH surge, as occurs in adult, cycling animals. Additionally, the expression pattern of PPARdelta mRNA indicates that it may be involved in cellular functions involved with maintaining basal ovarian function, whereas PPARalpha may play a role in lipid metabolism in the theca and stroma.