Follicle-Stimulating Hormone Glycosylation Variants Distinctly Modulate Pre-antral Follicle Growth and Survival.

Follicle-stimulating hormone (FSH) is a key endocrine regulator of ovarian function. FSH is secreted as 2 macroglycosylation variants: partially glycosylated FSH (FSH21/18) and fully glycosylated FSH (FSH24). FSH21/18 is more potent than FSH24 at binding to and activating the FSH receptor (R). The ratio of FSH21/18:FSH24 has been shown to change with age, with FSH21/18 predominant at reproductive prime, and FSH24 predominant during perimenopause/menopause. How these FSH glycosylation variants modulate ovarian follicle functions remains largely unknown. The aim of this study was to investigate the effect of FSH glycosylation variants of pre-antral follicle function. Pre-antral follicles were isolated from 3- to 5-week-old C57BL/6 mice and treated ±10 ng/mL FSH21/18, FSH24, a ratio of 80:20 FSH21/18:FSH24 (to mimic reproductive prime), 50:50 FSH21/18:FSH24 (perimenopause), or 20:80 FSH21/18:FSH24 (menopause) for up to 96 hours. FSH21/18 and 80:20 FSH21/18:FSH24 increased follicle growth, in comparison with control, contrasting with FSH24 and 20:80 FSH21/18:FSH24. Survival rates were decreased in follicles treated with FSH24 or 20:80 FSH21/18:FSH24, with follicles undergoing basement membrane rupture and oocyte extrusion, increased Caspase3 gene and protein expression, and decreased markers of cell proliferation in FSH24 or 20:80 FSH21/18:FSH24-treated follicles. Moreover, this correlated with differential regulation of key genes modulating follicular functions. Pharmacological inhibitors of key FSH signal pathways suggests FSH21/18 and FSH24 initiate different FSHR signal pathway activation, which may determine their differential effects on follicle growth and survival. These data suggest that the nature of FSH glycosylation modulates the follicular cellular environment to regulate follicle growth and survival and may underpin the increasing ovarian resistance to FSH observed during aging.

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.

Primary cilium-mediated MSC mechanotransduction is dependent on Gpr161 regulation of hedgehog signalling.

The benefits of physical loading to skeletal mass are well known. The primary cilium has emerged as an important organelle in bone mechanobiology/mechanotransduction, particularly in mesenchymal stem/stromal cells, yet the molecular mechanisms of cilium mechanotransduction are poorly understood. In this study, we demonstrate that Gpr161 is a mechanoresponsive GPCR, that localises to the cilium, and is involved in fluid shear-induced cAMP signalling and downstream osteogenesis. This Gpr161-mediated mechanotransduction is dependent on IFT88/cilium and may act through adenylyl cyclase 6 (AC6) to regulate cAMP and MSC osteogenesis. Moreover, we demonstrate that Hh signalling is positively associated with osteogenesis and that Hh gene expression is mechanically regulated and required for loading-induced osteogenic differentiation through a mechanism that involves IFT88, Gpr161, AC6, and cAMP. Therefore, we have delineated a molecular mechanism of MSC mechanotransduction which likely occurs at the cilium, leading to MSC osteogenesis, highlighting novel mechanotherapeutic targets to enhance osteogenesis.

Loss of Adenylyl Cyclase 6 in Leptin Receptor-Expressing Stromal Cells Attenuates Loading-Induced Endosteal Bone Formation.

Bone marrow stromal/stem cells represent a quiescent cell population that replenish the osteoblast bone-forming cell pool with age and in response to injury, maintaining bone mass and repair. A potent mediator of stromal/stem cell differentiation in vitro and bone formation in vivo is physical loading, yet it still remains unclear whether loading-induced bone formation requires the osteogenic differentiation of these resident stromal/stem cells. Therefore, in this study, we utilized the leptin receptor (LepR) to identify and trace the contribution of bone marrow stromal cells to mechanoadaptation of bone in vivo. Twelve-week-old Lepr-cre;tdTomato mice were subjected to compressive tibia loading with an 11 N peak load for 40 cycles, every other day for 2 weeks. Histological analysis revealed that Lepr-cre;tdTomato+ cells arise perinatally around blood vessels and populate bone surfaces as lining cells or osteoblasts before a percentage undergo osteocytogenesis. Lepr-cre;tdTomato+ stromal cells within the marrow increase in abundance with age, but not following the application of tibial compressive loading. Mechanical loading induces an increase in bone mass and bone formation parameters, yet does not evoke an increase in Lepr-cre;tdTomato+ osteoblasts or osteocytes. To investigate whether adenylyl cyclase-6 (AC6) in LepR cells contributes to this mechanoadaptive response, Lepr-cre;tdTomato mice were further crossed with AC6 fl/fl mice to generate a LepR+ cell-specific knockout of AC6. These Lepr-cre;tdTomato;AC6 fl/fl animals have an attenuated response to compressive tibia loading, characterized by a deficient load-induced osteogenic response on the endosteal bone surface. This, therefore, shows that Lepr-cre;tdTomato+ cells contribute to short-term bone mechanoadaptation. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

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.

Genomic identification, expression profiling, and functional characterization of CatSper channels in the bovine.

Cation channels of sperm (CatSper) are sperm-specific calcium channels with identified roles in the regulation of sperm function in humans, mice, and horses. We sought to employ a comparative genomics approach to identify conserved CATSPER genes in the bovine genome, and profile their expression in reproductive tissue. We hypothesized that CATSPER proteins expressed in bull testicular tissue mediates sperm hyperactivation and their rheotactic response in the reproductive tract of the cow. Bioinformatic analysis identified all four known CATSPER genes (CATSPER 1-4) in the bovine genome, and profiling by quantitative real-time polymerase chain reaction identified site-specific variation in messenger ribonucleic acid (mRNA) expression for all four genes along the reproductive tract of the bull. Using a novel antibody against CATSPER 1, protein expression was confirmed and localized to the principal piece of bull sperm, in agreement with what has been reported in other species. Subsequent treatment of bull sperm with either the calcium chelator ethylene glycol tetraacetic acid; mibefradil, a specific blocker of CatSper channels in human sperm; or CATSPER1 antibody all significantly inhibited caffeine-induced hyperactivation and the rheotactic response, supporting the concept that the calcium influx occurs via CatSper channels. Taken together, the work here provides novel insights into expression and function of CatSper channels in bull testicular tissue and in the function of ejaculated sperm.

Comparative genomic identification and expression profiling of a novel ?-defensin gene cluster in the equine reproductive tract.

?-defensins are small cationic proteins with potent immunoregulatory and antimicrobial activity. The number of genes encoding these peptides varies significantly between and within species but they have not been extensively characterised in the horse. Here, we describe a systematic search of the Equus caballus genome that identified a cluster of novel ?-defensin genes on Chromosome 22, which is homologous to a cluster on bovine Chromosome 13. Close genomic matches were found for orthologs of 13 of the bovine genes, which were named equine ?-defensins (eBD) 115, eBD116, eBD117, eBD119, eBD120, eBD122a, eBD123, eBD124, eBD125, eBD126, eBD127, eBD129 and eBD132. As expression of the homologous cluster in cattle was limited to the reproductive tract, tissue sections were obtained from the testis, caput, corpus and cauda epididymis and the vas deferens of three stallions and from the ovary, oviduct, uterine horn, uterus, cervix and vagina of three mares. Using a quantitative real-time polymerase chain reaction approach, each of the novel ?-defensin genes showed distinct region-specific patterns of expression. Preferential expression in the caput epididymis of these novel defensins in the stallion and in the oviduct in the mare suggests a possible role in immunoprotection of the equine reproductive tract or in fertility.