Hypo-glycosylated hFSH drives ovarian follicular development more efficiently than fully-glycosylated hFSH: enhanced transcription and PI3K and MAPK signaling.

STUDY QUESTION: Does hypo-glycosylated human recombinant FSH (hFSH18/21) have greater in vivo bioactivity that drives follicle development in vivo compared to fully-glycosylated human recombinant FSH (hFSH24)? SUMMARY ANSWER: Compared with fully-glycosylated hFSH, hypo-glycosylated hFSH has greater bioactivity, enabling greater follicular health and growth in vivo, with enhanced transcriptional activity, greater activation of receptor tyrosine kinases (RTKs) and elevated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling. WHAT IS KNOWN ALREADY: Glycosylation of FSH is necessary for FSH to effectively activate the FSH receptor (FSHR) and promote preantral follicular growth and formation of antral follicles. In vitro studies demonstrate that compared to fully-glycosylated recombinant human FSH, hypo-glycosylated FSH has greater activity in receptor binding studies, and more effectively stimulates the PKA pathway and steroidogenesis in human granulosa cells. STUDY DESIGN, SIZE, DURATION: This is a cross-sectional study evaluating the actions of purified recombinant human FSH glycoforms on parameters of follicular development, gene expression and cell signaling in immature postnatal day (PND) 17 female CD-1 mice. To stimulate follicle development in vivo, PND 17 female CD-1 mice (n = 8-10/group) were treated with PBS (150 µl), hFSH18/21 (1 µg/150 µl PBS) or hFSH24 (1 µg/150 µl PBS) by intraperitoneal injection (i.p.) twice daily (8:00 a.m. and 6:00 p.m.) for 2 days. Follicle numbers, serum anti-Müllerian hormone (AMH) and estradiol levels, and follicle health were quantified. PND 17 female CD-1 mice were also treated acutely (2 h) in vivo with PBS, hFSH18/21 (1 µg) or hFSH24 (1 µg) (n = 3-4/group). One ovary from each mouse was processed for RNA sequencing analysis and the other ovary processed for signal transduction analysis. An in vitro ovary culture system was used to confirm the relative signaling pathways. PARTICIPANTS/MATERIALS, SETTING, METHODS: The purity of different recombinant hFSH glycoforms was analyzed using an automated western blot system. Follicle numbers were determined by counting serial sections of the mouse ovary. Real-time quantitative RT-PCR, western blot and immunofluorescence staining were used to determine growth and apoptosis markers related with follicle health. RNA sequencing and bioinformatics were used to identify pathways and processes associated with gene expression profiles induced by acute FSH glycoform treatment. Analysis of RTKs was used to determine potential FSH downstream signaling pathways in vivo. Western blot and in vitro ovarian culture system were used to validate the relative signaling pathways. MAIN RESULTS AND THE ROLE OF CHANCE: Our present study shows that both hypo- and fully-glycosylated recombinant human FSH can drive follicular growth in vivo. However, hFSH18/21 promoted development of significantly more large antral follicles compared to hFSH24 (P < 0.01). In addition, compared with hFSH24, hFSH18/21 also promoted greater indices of follicular health, as defined by lower BAX/BCL2 ratios and reduced cleaved Caspase 3. Following acute in vivo treatment with FSH glycoforms RNA-sequencing data revealed that both FSH glycoforms rapidly induced ovarian transcription in vivo, but hypo-glycosylated FSH more robustly stimulated Gαs and cAMP-mediated signaling and members of the AP-1 transcription factor complex. Moreover, hFSH18/21 treatment induced significantly greater activation of RTKs, PI3K/AKT and MAPK/ERK signaling compared to hFSH24. FSH-induced indices of follicle growth in vitro were blocked by inhibition of PI3K and MAPK. LARGE SCALE DATA: RNA sequencing of mouse ovaries. Data will be shared upon reasonable request to the corresponding author. LIMITATIONS, REASONS FOR CAUTION: The observations that hFSH glycoforms have different bioactivities in the present study employing a mouse model of follicle development should be verified in nonhuman primates. The gene expression studies reflect transcriptomes of whole ovaries. WIDER IMPLICATIONS OF THE FINDINGS: Commercially prepared recombinant human FSH used for ovarian stimulation in human ART is fully-glycosylated FSH. Our findings that hypo-glycosylated hFSH has greater bioactivity enabling greater follicular health and growth without exaggerated estradiol production in vivo, demonstrate the potential for its development for application in human ART. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by NIH 1P01 AG029531, NIH 1R01 HD 092263, VA I01 BX004272, and the Olson Center for Women's Health. JSD is the recipient of a VA Senior Research Career Scientist Award (1IK6 BX005797). This work was also partially supported by National Natural Science Foundation of China (No. 31872352). The authors declared there are no conflicts of interest.

Human bone marrow stem/stromal cell osteogenesis is regulated via mechanically activated osteocyte-derived extracellular vesicles.

Bone formation or regeneration requires the recruitment, proliferation, and osteogenic differentiation of stem/stromal progenitor cells. A potent stimulus driving this process is mechanical loading. Osteocytes are mechanosensitive cells that play fundamental roles in coordinating loading-induced bone formation via the secretion of paracrine factors. However, the exact mechanisms by which osteocytes relay mechanical signals to these progenitor cells are poorly understood. Therefore, this study aimed to demonstrate the potency of the mechanically stimulated osteocyte secretome in driving human bone marrow stem/stromal cell (hMSC) recruitment and differentiation, and characterize the secretome to identify potential factors regulating stem cell behavior and bone mechanobiology. We demonstrate that osteocytes subjected to fluid shear secrete a distinct collection of factors that significantly enhance hMSC recruitment and osteogenesis and demonstrate the key role of extracellular vesicles (EVs) in driving these effects. This demonstrates the pro-osteogenic potential of osteocyte-derived mechanically activated extracellular vesicles, which have great potential as a cell-free therapy to enhance bone regeneration and repair in diseases such as osteoporosis.

Mechanistic insight into how gonadotropin hormone receptor complexes direct signaling†.

Gonadotropin hormones and their receptors play a central role in the control of male and female reproduction. In recent years, there has been growing evidence surrounding the complexity of gonadotropin hormone/receptor signaling, with it increasingly apparent that the Gαs/cAMP/PKA pathway is not the sole signaling pathway that confers their biological actions. Here we review recent literature on the different receptor-receptor, receptor-scaffold, and receptor-signaling molecule complexes formed and how these modulate and direct gonadotropin hormone-dependent intracellular signal activation. We will touch upon the more controversial issue of extragonadal expression of FSHR and the differential signal pathways activated in these tissues, and lastly, highlight the open questions surrounding the role these gonadotropin hormone receptor complexes and how this will shape future research directions.

Management of Herniated Lumbar Disk Disease and Cauda Equina Syndrome in Pregnancy.

Lower back pain is a commonly reported symptom during pregnancy. However, herniated lumbar disk disease is an uncommon cause for such pain. Cauda equina syndrome (CES) during pregnancy is a rare clinical scenario. This review highlights the epidemiology, diagnostic and treatment strategies, and challenges encountered when managing herniated lumbar disk disease and CES in pregnancy. Magnetic resonance imaging is the diagnostic modality of choice. Nonoperative treatment strategies are successful in the vast majority of cases in patients with a herniated disk in the absence of CES. CES and progressive neurological deficits remain absolute indications for surgical intervention regardless of gestational age. For such patients or those with debilitating symptoms refractory to nonoperative treatment strategies, surgery has been demonstrated to be safe in the pregnant patient population. However, surgery should be performed with obstetric and midwifery support should complications occur to the fetus.

Physiological cyclic hydrostatic pressure induces osteogenic lineage commitment of human bone marrow stem cells: a systematic study.

BACKGROUND: Physical loading is necessary to maintain bone tissue integrity. Loading-induced fluid shear is recognised as one of the most potent bone micromechanical cues and has been shown to direct stem cell osteogenesis. However, the effect of pressure transients, which drive fluid flow, on human bone marrow stem cell (hBMSC) osteogenesis is undetermined. Therefore, the objective of the study is to employ a systematic analysis of cyclic hydrostatic pressure (CHP) parameters predicted to occur in vivo on early hBMSC osteogenic responses and late-stage osteogenic lineage commitment. METHODS: hBMSC were exposed to CHP of 10 kPa, 100 kPa and 300 kPa magnitudes at frequencies of 0.5 Hz, 1 Hz and 2 Hz for 1 h, 2 h and 4 h of stimulation, and the effect on early osteogenic gene expression of COX2, RUNX2 and OPN was determined. Moreover, to decipher whether CHP can induce stem cell lineage commitment, hBMSCs were stimulated for 4 days for 2 h/day using 10 kPa, 100 kPa and 300 kPa pressures at 2 Hz frequency and cultured statically for an additional 1-2 weeks. Pressure-induced osteogenesis was quantified based on ATP release, collagen synthesis and mineral deposition. RESULTS: CHP elicited a positive, but variable, early osteogenic response in hBMSCs in a magnitude- and frequency-dependent manner, that is gene specific. COX2 expression elicited magnitude-dependent effects which were not present for RUNX2 or OPN mRNA expression. However, the most robust pro-osteogenic response was found at the highest magnitude (300 kPa) and frequency regimes (2 Hz). Interestingly, long-term mechanical stimulation utilising 2 Hz frequency elicited a magnitude-dependent release of ATP; however, all magnitudes promoted similar levels of collagen synthesis and significant mineral deposition, demonstrating that lineage commitment is magnitude independent. This therefore demonstrates that physiological levels of pressures, as low as 10 kPa, within the bone can drive hBMSC osteogenic lineage commitment. CONCLUSION: Overall, these findings demonstrate an important role for cyclic hydrostatic pressure in hBMSCs and bone mechanobiology, which should be considered when studying pressure-driven fluid shear effects in hBMSCs mechanobiology. Moreover, these findings may have clinical implication in terms of bioreactor-based bone tissue engineering strategies.

Mesenchymal stem cell mechanotransduction is cAMP dependent and regulated by adenylyl cyclase 6 and the primary cilium.

Mechanical loading is a potent stimulus of bone adaptation, requiring the replenishment of the osteoblast from a progenitor population. One such progenitor is the mesenchymal stem cell (MSC), which undergoes osteogenic differentiation in response to oscillatory fluid shear. Yet, the mechanism mediating stem cell mechanotransduction, and thus the potential to target this therapeutically, is poorly understood. In this study, we demonstrate that MSCs utilise cAMP as a second messenger in mechanotransduction, which is required for flow-mediated increases in osteogenic gene expression. Furthermore, we demonstrate that this mechanosignalling is dependent on the primary cilium and the ciliary localised adenylyl cyclase 6. Finally, we also demonstrate that this mechanotransduction mechanism can be targeted therapeutically to enhance cAMP signalling and early osteogenic signalling, mimicking the beneficial effect of physical loading. Our findings therefore demonstrate a novel mechanism of MSC mechanotransduction that can be targeted therapeutically, demonstrating a potential mechanotherapeutic for bone-loss diseases such as osteoporosis.This article has an associated First Person interview with the first author of the paper.

TRPV4-mediates oscillatory fluid shear mechanotransduction in mesenchymal stem cells in part via the primary cilium.

Skeletal homeostasis requires the continued replenishment of the bone forming osteoblast from a mesenchymal stem cell (MSC) population, a process that has been shown to be mechanically regulated. However, the mechanisms by which a biophysical stimulus can induce a change in biochemical signaling, mechanotransduction, is poorly understood. As a precursor to loading-induced bone formation, deciphering the molecular mechanisms of MSC osteogenesis is a critical step in developing novel anabolic therapies. Therefore, in this study we characterize the expression of the mechanosensitive calcium channel Transient Receptor Potential subfamily V member 4 (TRPV4) in MSCs and demonstrate that TRPV4 localizes to areas of high strain, specifically the primary cilium. We demonstrate that TRPV4 is required for MSC mechanotransduction, mediating oscillatory fluid shear induced calcium signaling and early osteogenic gene expression. Furthermore, we demonstrate that TRPV4 can be activated pharmacologically eliciting a response that mirrors that seen with mechanical stimulation. Lastly, we show that TRPV4 localization to the primary cilium is functionally significant, with MSCs with defective primary cilia exhibiting an inhibited osteogenic response to TRPV4 activation. Collectively, this data demonstrates a novel mechanism of stem cell mechanotransduction, which can be targeted therapeutically, and further highlights the critical role of the primary cilium in MSC biology.