Biology subjects

Uliasz, T. F.

Publications and source records attributed to Uliasz, T. F..

4 recordsLinked to original sources

bioRxiv2023

Phosphatases modified by LH signaling in rat ovarian follicles and their role in regulating the NPR2 guanylyl cyclase

In response to luteinizing hormone, multiple proteins in rat and mouse granulosa cells are rapidly dephosphorylated, but the responsible phosphatases remain to be identified. Because the phosphorylation state of phosphatases can regulate their interaction with substrates, we searched for phosphatases that might function in LH signaling by using quantitative mass spectrometry. We identified all proteins in rat ovarian follicles whose phosphorylation state changed detectably in response to a 30-minute exposure to LH, and within this list, identified protein phosphatases or phosphatase regulatory subunits that showed changes in phosphorylation. Phosphatases in the PPP family were of particular interest because of their requirement for dephosphorylating the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase in the granulosa cells, which triggers oocyte meiotic resumption. Among the PPP family regulatory subunits, PPP1R12A and PPP2R5D showed the largest increases in phosphorylation, with 4-10 fold increases in signal intensity on several sites. Although follicles from mice in which these phosphorylations were prevented by serine-to-alanine mutations in either Ppp1r12a or Ppp2r5d showed normal LH-induced NPR2 dephosphorylation, these regulatory subunits and others could act redundantly to dephosphorylate NPR2. Our identification of phosphatases and other proteins whose phosphorylation state is rapidly modified by LH provides clues about multiple signaling pathways in ovarian follicles. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/544636v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@2622cdorg.highwire.dtl.DTLVardef@1b6eef7org.highwire.dtl.DTLVardef@102da9borg.highwire.dtl.DTLVardef@5e3221_HPS_FORMAT_FIGEXP M_FIG C_FIG Summary sentenceQuantitative mass spectrometric analysis of phosphatases whose phosphorylation state is rapidly modified by luteinizing hormone provides clues about how LH signaling dephosphorylates NPR2 as well as a resource for future studies.

cell biology↗

bioRxiv2020

The phosphatase inhibitor LB-100 acts synergistically with the NPR2 agonist BMN-111 to improve bone growth

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations in the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase both result in decreased production of cyclic GMP (cGMP) in chondrocytes and severe short stature, causing achondroplasia (ACH) and acrosomelic dysplasia type Maroteaux, respectively. Previously we showed that an NPR2 agonist BMN-111 (vosoritide) increases bone growth in mice mimicking ACH (Fgfr3Y367C/+), and that in control growth plate chondrocytes, FGFR3 signaling decreases NPR2 activity by dephosphorylating the NPR2 protein. Here we tested whether a phosphatase inhibitor (LB-100) could enhance bone growth in ACH. In ex vivo imaging experiments using a FRET sensor to measure cGMP production in chondrocytes of living tibias from newborn mice, LB-100 counteracts the FGF-induced dephosphorylation and inactivation of NPR2. In ex vivo experiments with Fgfr3Y367C/+ mice, LB-100 in combination with BMN-111 increases the rate of femur growth by [~]25% vs BMN-111 alone, restores chondrocyte terminal differentiation, increases the proliferative growth plate area of the femur, and reduces the activity of the MAP kinase pathway. Our results provide a proof of concept that a phosphatase inhibitor could be used together with an NPR2 agonist to enhance cGMP production as a therapy for ACH. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/288589v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1d0f5d3org.highwire.dtl.DTLVardef@1bb0724org.highwire.dtl.DTLVardef@1d206d1org.highwire.dtl.DTLVardef@1bd9ad7_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2020

Testosterone use in female mice does not impair fertilizability of eggs: Implications for the fertility care of transgender males

STUDY QUESTIONDoes testosterone use in females affect reproductive potential, particularly with regard to the production of fertilizable gametes? SUMMARY ANSWERTestosterone cypionate injections given to post-pubertal female mice caused virilization and ovaries were smaller than control ovaries, but ovaries were still responsive to hormonal stimulation and produced fertilizable eggs when superovulated. WHAT IS KNOWN ALREADYStudies to examine the effects of testosterone on reproductive potential in transgender males are lacking. Recently, a model was developed that simulates many aspects of testosterone use in transgender males in order to look at reproductive effects of testosterone in female mice. This study found masculinizing effects on the mice but did not find significant deficits on the number of ovarian follicles; however, effects of testosterone use on ovarian stimulation and fertilizability of oocytes were not investigated. STUDY DESIGN, SIZE, DURATIONA total of 66, 6-week-old Hsd:NSA(CF-1) female mice and 6 Hsd:ICR (CD-1) mice were used for this study. Mice were injected subcutaneously with 400 g testosterone cypionate or sesame oil once a week for 6 weeks and were either sacrificed a week after the 6th injection (active exposure group), or were sacrificed 6-7 weeks after the final testosterone injection (washout group). PARTICIPANTS/MATERIALS, SETTING, METHODSBoth active exposure and washout groups were further subdivided into 3 groups: unstimulated, eCG-stimulated, or eCG/hCG-stimulated. eCG-stimulated mice were sacrificed 44-48 hrs after eCG injection. eCG/hCG-stimulated mice were injected with eCG, followed 48 hrs later with hCG. Mice were sacrificed [~]13-18 hrs after the hCG injection. Data collected included daily vaginal cytology, terminal hormone levels and ovary weights, ovarian histology, number of oocytes/eggs collected in each group, and cleavage to the 2-cell stage following in vitro fertilization. MAIN RESULTS AND THE ROLE OF CHANCETestosterone cypionate-treated mice had testosterone levels elevated to the level of male mice and ceased cycling. Ovaries were significantly smaller in testosterone-treated mice, but they contained normal cohorts of follicles and responded to gonadotropin stimulation by ovulating similar numbers of eggs that fertilized and cleaved in vitro. LIMITATIONS, REASONS FOR CAUTIONOur model treated female mice for only 6 weeks, whereas many transgender men use testosterone for many years before considering biological children. Importantly, a mouse system may not perfectly simulate human reproductive physiology. WIDER IMPLICATIONS OF THE FINDINGSThe current standard of care for transgender men who desire biological children is to cease testosterone therapy prior to ovarian stimulation, but the necessity for stopping testosterone is not known. Our model demonstrates that it is possible for testosterone-suppressed ovaries to respond to gonadotropic stimulation by producing and ovulating fertilizable eggs, thereby obviating the need for testosterone cessation prior to ovarian stimulation. In time, these results may provide insights for future clinical trials of fertility treatment options for transgender men.

physiology↗

bioRxiv2019

SNAP23 is required for the maintenance of meiotic arrest and cortical granule exocytosis in mouse oocytes

Mammalian oocytes are stored in the ovary for prolonged periods, arrested in meiotic prophase. During this period, their plasma membranes are constantly being recycled by endocytosis and exocytosis. However, the function of this membrane turnover is unknown. Here, we investigated the requirement for exocytosis in the maintenance of meiotic arrest. Using a newly developed method for rapidly and specifically depleting proteins in oocytes, we have identified the SNARE protein, SNAP23, to be required for meiotic arrest. Degradation of SNAP23 causes premature meiotic resumption in follicle-enclosed oocytes. The reduction in SNAP23 is associated with loss of gap junction communication between the oocyte and surrounding follicle cells. Reduction of SNAP23 protein also inhibits cortical granule exocytosis in response to a Ca2+ stimulus, as measured by lectin staining and cleavage of ZP2. Our results show an essential role for SNAP23 in two key processes that occur in mouse oocytes and eggs. SummaryThe SNARE protein, SNAP23, is required to maintain gap junction communication between the oocyte and follicle cells that is needed to maintain oocyte meiotic arrest, as well as for cortical granule exocytosis at fertilization.

cell biology↗