Nucleolar Channel System Research Articles

Extruded Nucleoli of Human Dental Pulp Cells.

Background and Objectives: The dental pulp stem cells are highly proliferative and can differentiate into various cell types, including endothelial cells. We aimed to evaluate the ultrastructural characteristics of the human dental pulp cells of the permanent frontal teeth. Materials and Methods: Human adult bioptic dental pulp was collected from n = 10 healthy frontal teeth of five adult patients, prior to prosthetic treatments for aesthetic purposes. Tissues were examined under transmission electron microscopy. Results: We identified cells with a peculiar trait: giant nucleoli resembling intranuclear endoplasmic reticulum, which mimicked extrusion towards the cytoplasm. These were either partly embedded within the nuclei, the case in which their adnuclear side was coated by marginal heterochromatin and the abnuclear side was coated by a thin rim of ribosomes, or were apparently isolated from the nuclei, while still being covered by ribosomes. Conclusions: Similar electron microscopy features were previously reported in the human endometrium, as nucleolar channel system; or R-Rings induced by Nopp140. To our knowledge, this is the first report of extruded nucleolar structure in the dental pulp. Moreover, the aspect of giant extruded nucleoli was not previously reported in any human cell type, although similar evidence was gathered in other species as well as in plants.

A Transient Mystery: Nucleolar Channel Systems.

The nucleus is a complex organelle with functions beyond being a simple repository for genomic material. For example, its actions in biomechanical sensing, protein synthesis, and epigenomic regulation showcase how the nucleus integrates multiple signaling modalities to intricately regulate gene expression. This innate dynamism is underscored by subnuclear components that facilitate these roles, with elementsof the nucleoskeleton, phase-separated nuclear bodies, and chromatin safeguarding by nuclear envelope proteins providingexamplesof this functional diversity. Among these, one of the lesser characterized nuclear organelles is the nucleolar channel system (NCS), first reported several decades ago inhuman endometrial biopsies. This tubular structure, believed to be derived from the inner nuclear membrane of the nuclear envelope, was first observed in secretory endometrial cells during a specific phase of the menstrual cycle. Reported as a consistent, yet transient, nuclear organelle, current interpretations of existing data suggest that it serves as a marker of a window for optimal implantation. In spite of this available data, the NCS remains incompletely characterized structurally and functionally, due in part to its transient spatial and temporal expression. As a further complication, evidence exists showing NCS expression in fetal tissue, suggesting that it may not act exclusively as a marker of uterine receptivity, but rather as a hormone sensor sensitive to estrogen and progesterone ratios. To gain a better understanding of the NCS, current technologies can be applied to profile rare cell populations or capture transient structural dynamics, for example, at a level of sensitivity and resolution not previously possible. Moving forward, advanced characterization of the NCS will shed light on an uncharacterized aspect of reproductive physiology, with the potential to refine assisted reproductive techniques.

Presence of endometrial nucleolar channel systems at the time of frozen embryo transfer in hormone replacement cycles with successful implantation

To detect nucleolar channel systems (NCSs) in cells in endometrial aspirations obtained immediately before embryo transfer during blastocyst hormone replacement therapy-frozen embryo transfer (HRT-FET) cycles without affecting implantation. Prospective case series. University-affiliated fertility clinic. Five patients who underwent an HRT-FET cycle consented to lower uterine segment aspiration using an open-tip embryo transfer catheter during a routine mock transfer performed immediately before embryo transfer. Exfoliated cells in the aspirated endometrial secretions were analyzed for the presence of NCSs using indirect immunofluorescence and, in one case, electron microscopy for unambiguous identification. On the basis of a previous study, positive NCS status was defined as the presence of NCSs in at least 3 endometrial epithelial cells (EECs). The effect of endometrial aspiration on implantation and pregnancy outcomes was assessed. Biochemical pregnancy, as evidenced by positive β-human chorionic gonadotropin, was seen in 5 of 5 patients, and clinical pregnancy was seen in 2 of 5 patients. NCSs were detected in exfoliated EECs of uterine secretions in 4 of 5 patient samples and could not be unequivocally identified in 1 of 5 patient samples, which was designated as indeterminate. This is the first report of NCS detection in HRT-FET cycles in the absence of follicular development and ovulation. NCS status can be determined in exfoliated EECs of uterine secretions obtained at the time of embryo transfer while maintaining implantation. Our study furthers the goal of establishing whether individualized point of care testing of NCS status in HRT-FET cycles can determine optimal endometrial receptivity and improve pregnancy outcomes.

Identification of nucleolar channel systems (NCSs) in endometrial secretions at the time of frozen embryo transfer in artificial cycles with successful implantation

Identification of nucleolar channel systems (NCSs) in endometrial secretions at the time of frozen embryo transfer in artificial cycles with successful implantation

Status of nucleolar channel systems in uterine secretions accurately reflects their prevalence—a marker for the window of implantation—in simultaneously obtained endometrial biopsies

To assess whether nucleolar channel systems (NCSs) can be detected in exfoliated endometrial epithelial cells (EECs) of uterine secretions and whether such noninvasively determined NCS status is associated with significant NCS prevalence in simultaneously obtained endometrial biopsies. Prospective study (December 2015-February 2017). University-affiliated and private fertility clinics. Luteal-phase patients of reproductive age requiring endometrial biopsy for medical indications. Uterine secretion aspiration before endometrial biopsy. Cells in uterine secretions were spun onto slides and fixed. NCSs were identified and quantified in cells and paraffin-embedded tissue sections by indirect immunofluorescence. Comparison of NCS status of uterine secretions with NCS prevalence in biopsies. Based on NCS detection, uterine secretions were assigned a status of NCS-positive (n = 15) or NCS-negative (n = 7). NCS prevalence in biopsies was expressed as a percentage of NCSs per EECs. NCSs can be detected in exfoliated EECs of uterine secretions. Median NCS prevalence in endometrial biopsies from patients with NCS-positive secretions was 41.9% (interquartile range [IQR], 21.1-53.9) versus 2.0% (IQR, 0-6.9) when secretions were NCS-negative. The NCS status of secretions identified a significant difference in NCS prevalence of simultaneously obtained biopsies. NCS status of secretions accurately reflects NCS prevalence of biopsies, a marker for the implantation window. As secretion aspiration is compatible with same-day ET, our study provides proof of principle for a minimally invasive approach to determine endometrial receptivity for timing frozen ET.

Timing the window of implantation by nucleolar channel system prevalence matches the accuracy of the endometrial receptivity array

To test if nucleolar channel system (NCS) prevalence matches the accuracy of the endometrial receptivity array (ERA) for identification of the window of endometrial receptivity. Comparative retrospective study, May 2008-May 2012. University-affiliated infertility clinic. Forty-nine healthy oocyte donors, regularly cycling, aged 20-34 years with a body mass index of 19-25 kg/m2. Endometrial biopsies were collected throughout the menstrual cycle. All samples underwent transcriptomic signature identification by ERA testing (performed in a prior study) and quantification of NCS prevalence by using indirect immunofluorescence (performed in the present study). Concordance of ERA results determining the window of implantation with NCS prevalence was statistically analyzed using the kappa index. Based on dating according to the luteinizing hormone surge, specimens were dichotomized into receptive (n=24) and nonreceptive (n=25). The NCS prevalence was expressed as percentage of NCSs per endometrial epithelial cells in each endometrial biopsy. Concordance of ERA and NCS dating vs. luteinizing hormone yielded comparable kappa indices of 0.878 and 0.836, respectively. Direct comparison of ERA and NCS dating resulted in a kappa index of 0.796. Prevalence of NCS identifies the window of endometrial receptivity previously identified by their transcriptomic signature using the ERA.

Progesterone Threshold Determines Nucleolar Channel System Formation in Human Endometrium

Nucleolar channel systems (NCSs), micron-sized organelles specific to nuclei of human endometrial epithelial cells (EECs), are robust markers of the midluteal phase under the apparent control of progesterone. To gain further insight into the role of progesterone in NCS formation, we quantitatively assessed their sensitivity to oral contraceptive pills (OCPs) using immunofluorescence-based detection of NCSs. Comparison of endometrial biopsies and serum progesterone levels on cycle day (CD) 10 and 20 (LH +6/7) of 6 naturally cycling women and 6 OCP users demonstrated that OCPs interfered with NCS formation on CD20, their natural peak presence. Although this confirmed prior observation based on electron microscopic sampling, OCPs unexpectedly induced limited but distinct amounts of NCSs already on CD10, when they are never present in natural cycles. Thus, OCPs can cause secretory changes in the endometrium during the proliferative phase. In a novel finding, robust NCS formation on CD20 was dependent on a 4 ng/mL progesterone threshold but did not correlate linearly with serum progesterone levels. Given the threshold being close to that serving as evidence for ovulation, NCSs can serve as ovulation markers.

Premature formation of nucleolar channel systems indicates advanced endometrial maturation following controlled ovarian hyperstimulation.

Is there a shift in the timing of nucleolar channel system (NCS) formation following controlled ovarian hyperstimulation (COH)? NCSs appear prematurely following COH compared with natural cycles. During natural cycles, NCSs of endometrial epithelial cell (EEC) nuclei are exclusively present during the window of implantation and are uniformly distributed throughout the upper endometrial cavity. Prospective two-cohort study. Cohorts I and II each consisted of seven volunteers for the duration of three menstrual study cycles that were separated by at least one wash-out or rest cycle, between December 2008 and May 2012. Participants were recruited from a pool of healthy oocyte donors. Consecutive endometrial biopsies were obtained during the same luteal phase on cycle days (CD) 16, 20 and 26 for Cohort I, and on CD14, 22 and 24 for Cohort II, following random assignment to a natural cycle group, a COH cycle group (using a GnRH antagonist), or a COH cycle group receiving luteal phase hormonal supplementation (COH + S). The day of oocyte retrieval was designated CD14 in COH cycles and the day of the LH surge was designated CD13 in natural cycles. Prevalence of NCSs in the nuclei of EECs was quantified using indirect immunofluorescence with an antibody directed against a subset of related nuclear pore complex proteins that are major constituents of NCSs. Progesterone and estradiol levels were measured on the day of each endometrial biopsy. The natural cycle group exhibited peak NCS prevalence on CD20 [53.3%; interquartile range (IQR) 28.5-55.8], which rapidly declined on CD22 (11.8%; IQR 6.3-17.6), CD24 (2.5%; IQR 0.0-9.2) and CD26 (0.3%; IQR 0.0-3.5), and no NCSs on CD14 and 16 defining a short NCS window around CD20. In contrast, in COH and COH + S cycles, NCS prevalence was high already on CD16 (40.4%; IQR 22.6-53.4 and 35.6%; IQR 26.4-44.5, respectively; P = 0.001 compared with CD16 of the natural cycle group, Mann-Whitney), whereas no significant difference in NCS prevalence was detected on any of the other five CDs between the three groups (P > 0.05). The cohort size was small (n = 7) but was offset by the all-or-none presence of NCSs on CD16 in natural versus COH and COH + S cycles and the fact that each subject served as her own control. Premature appearance of NCSs and hence maturation of the endometrium following COH is consistent with previous studies based on histological dating but contradicts studies based on mRNA expression profiling, which reported a lag in endometrial maturation. However, this is the first study of this kind that is based on consecutive endometrial biopsies within the same cycle and that reports such clear-cut differences: no versus robust NCS presence on CD16. Our observation of advanced endometrial maturation following COH may contribute to the reduced implantation rates seen in fresh compared with frozen and donor IVF-embryo transfer cycles. Therefore, the NCS window could serve as a sensitive guide for timing of embryo transfer in frozen and donor cycles. The study was supported by the March of Dimes Birth Defects foundation (1-FY09-363 to U.T.M.); Ferring Pharmaceuticals, Parsippany, NJ; East Coast Fertility, Plainview, NY and the CMBG Training Program (T32 GM007491 to M.J.S.). We report no competing interests.

Premature formation of nucleolar channel systems indicates advanced endometrial maturation following controlled ovarian hyperstimulation

To evaluate the temporal appearance of nucleolar channel systems (NCSs) in the human secretory endometrium in natural cycles (Nat) and following ovarian hyperstimulation with (OH+S) and without (OH) vaginal progesterone (P4) (Endometrin; Ferring Pharmaceuticals). Is there a shift in NCS formation following controlled ovarian hyperstimulation (COH)? Prospective two-cohort study. Consecutive endometrial biopsies were obtained from healthy, oocyte donors on cycle days (CD) 16, 20 and 26 (cohort I, n=7), or CDs 14, 22 and 24 (cohort II, n=8) following random assignment to a Nat, OH or OH+S group, with at least one ‘wash-out’ cycle between groups. NCS prevalence in the nuclei of endometrial epithelial cells (EECs) was quantified (#NCSs/#EECs x 100, %) using indirect immunofluorescence with an antibody directed against certain nuclear pore complex proteins, that are major constituents of NCSs. Estradiol and P4 levels were measured on the day of each biopsy. Nat group exhibited peak NCS prevalence on CD20 (53%; interquartile range [IQR] 28.5-55.8), rapid decline on CD22, 24 and 26, and no NCSs on CD14 and 16. In contrast, after OH and OH+S, NCS prevalence was high already on CD16 (40.4%; IQR 22.6-53.4 and 35.6%; IQR 26.4-44.5, respectively; p=0.001, Mann-Whitney). No other significant differences were noted between the groups on any of the other five cycle days (p>0.1). In these healthy oocyte donors, NCS prevalence occurs when expected in the mid-luteal phase of natural cycles. In contrast, premature appearance of NCSs and hence maturation of the endometrium is observed following COH. This is the first study of this kind that is based on consecutive endometrial biopsies within the same cycle and that reports all-or-none data. Our observation of advanced endometrial maturation following COH may contribute to the reduced implantation success rates in fresh vs. frozen IVF-ET cycles.

Quantification of nucleolar channel systems: uniform presence throughout the upper endometrial cavity

To determine the prevalence of nucleolar channel systems (NCSs) by uterine region, applying continuous quantification. Prospective clinical study. Tertiary care academic medical center. Forty-two naturally cycling women who underwent hysterectomy for benign indications. NCS presence was quantified by a novel method in six uterine regions-fundus, left cornu, right cornu, anterior body, posterior body, and lower uterine segment (LUS)-with the use of indirect immunofluorescence. Percentage of endometrial epithelial cells (EECs) with NCSs per uterine region. NCS quantification was observer independent (intraclass correlation coefficient 0.96) and its intrasample variability low (coefficient of variation 0.06). Eleven of 42 hysterectomy specimens were midluteal, ten of which were analyzable with nine containing >5% EECs with NCSs in at least one region. The percentage of EECs with NCSs varied significantly between the LUS (6.1%; interquartile range [IQR] 3.0-9.9) and the upper five regions (16.9%; IQR 12.7-23.4), with fewer NCSs in the basal layer of the endometrium (17 ± 6%) versus the middle (46 ± 9%) and luminal layers (38 ± 9%) of all six regions. NCS quantification during the midluteal phase demonstrates uniform presence throughout the endometrial cavity, excluding the LUS, with a preference for the functional luminal layers. Our quantitative NCS evaluation provides a benchmark for future studies and further supports NCS presence as a potential marker for the window of implantation.

It’s time to pay attention to the endometrium

The physiologic importance of the endometrium in assisted reproductive technology is now receiving much research attention. The historical importance of ultrasound for determination of endometrial pattern, thickness and contractility is reviewed and speculation provided for a possible mechanism of the adverse effect of thin endometrium on implantation.

The nucleolar channel system reliably marks the midluteal endometrium regardless of fertility status: a fresh look at an old organelle

To determine whether nucleolar channel systems (NCSs) in the midluteal endometrium are associated with overall fertility status and/or with unexplained infertility. Retrospective and prospective clinical studies. Repository of stored specimens from prior multicenter study and private infertility center. Retrospective study that included 97 women (49 fertile couples, 48 infertile couples) who had been randomized for endometrial biopsy during the midluteal or late luteal phase. The prospective study included 78 women with a variety of infertility diagnoses. Endometrial biopsies were obtained and assessed for the presence of NCSs by indirect immunofluorescence. The presence of NCS was graded semiquantitatively and dichotomized as normal versus low or absent. Normal presence of NCS was significantly associated with the midluteal phase compared with the late luteal phase (80% vs. 29%). However, there was no association between presence of NCS and fertility status or between presence of NCS and unexplained infertility. Midluteal phase endometrium consistently forms NCSs regardless of fertility status, including unexplained infertility. This indicates a possible role for the NCS in initiating the window of endometrial receptivity. However, the consistent presence of NCSs across several different types of infertility challenges the likelihood that inadequate secretory transformation is a cause of infertility.

Shift in appearance of nucleolar channel systems in women following ovarian hyperstimulation with and without luteal hormonal supplementation

OBJECTIVE: To evaluate the temporal appearance of nucleolar channel systems (NCSs) in the human secretory endometrium in natural cycles (NC) and following ovarian hyperstimulation with (OH+S) and without (OH) vaginal progesterone (P4) (Endometrin; Ferring Pharmaceuticals). We hypothesized that NCSs might appear earlier or later following ovarian hyperstimulation (OvH).

Nuclear pore complex proteins mark the implantation window in human endometrium.

Nucleolar channel systems (NCSs) are membranous organelles appearing transiently in the epithelial cell nuclei of postovulatory human endometrium. Their characterization and use as markers for a healthy receptive endometrium have been limited because they are only identifiable by electron microscopy. Here we describe the light microscopic detection of NCSs using immunofluorescence. Specifically, the monoclonal nuclear pore complex antibody 414 shows that NCSs are present in about half of all human endometrial epithelial cells but not in any other cell type, tissue or species. Most nuclei contain only a single NCS of uniform 1 microm diameter indicating a tightly controlled organelle. The composition of NCSs is as unique as their structure; they contain only a subset each of the proteins of nuclear pore complexes, inner nuclear membrane, nuclear lamina and endoplasmic reticulum. Validation of our robust NCS detection method on 95 endometrial biopsies defines a 6-day window, days 19-24 (+/-1) of an idealized 28 day cycle, wherein NCSs occur. Therefore, NCSs precede and overlap with the implantation window and serve as potential markers of uterine receptivity. The immunodetection assay, combined with the hitherto underappreciated prevalence of NCSs, now enables simple screening and further molecular and functional dissection.

The nucleolar channel system of human endometrium is related to endoplasmic reticulum and R-rings.

The nucleolar channel system (NCS) is a well-established ultrastructural hallmark of the postovulation endometrium. Its transient presence has been associated with human fertility. Nevertheless, the biogenesis, composition, and function of these intranuclear membrane cisternae are unknown. Membrane systems with a striking ultrastructural resemblance to the NCS, termed R-rings, are induced in nuclei of tissue culture cells by overexpression of the central repeat domain of the nucleolar protein Nopp140. Here we provide a first molecular characterization of the NCS and compare the biogenesis of these two enigmatic organelles. Like the R-rings, the NCS consists of endoplasmic reticulum harboring the marker glucose-6-phosphatase. R-ring formation initiates at the nuclear envelope, apparently by a calcium-mediated Nopp140-membrane interaction, as supported by the calcium-binding ability of Nopp140, the inhibition of R-ring formation by calcium chelators, and the concentration of Nopp140 and complexed calcium in R-rings. Although biogenesis of the NCS may initiate similarly, the reduced presence of complexed calcium and Nopp140 suggests the involvement of additional factors.

412 Identification of pesticides sex-specific gonadal toxicity

While there are certain differences between biological responses to C-21 progestogens and the 19-norsteroids, precoital progestogens in general interfere with sperm transport, fertilization, and ovum transport. A 36 hour interval was required to achieve 100 per cent precoital contraceptive effectiveness in the rabbit. At marginal doses or timing, blastocyst degeneration may occur, and with large doses of progesterone at the time of coitus some teratogenic effects were noted. Postcoital estrogen is associated with lowering of plasma progesterone levels. Concomitant progesterone administartion, however, does not completely reverse the interceptive effect of estrogen. Direct effects on the endometrium include disintegration of the nucleolar channel system, retention of glycogen in endometrial glands, decreased alkaline phosphatase, and lowered endometrial carbonic anhydrase. The latter may be the basic mechanism of action.

Intranuclear endoplasmic reticulum induced by Nopp140 mimics the nucleolar channel system of human endometrium.

Exogenous expression of the characteristic repeat domain of the nucleolar chaperone Nopp140 induces the formation of intranuclear structures, termed R-rings. Here, the R-rings are identified as extensive stacks of membrane cisternae in the otherwise membrane-free nucleus. They consist of bona fide endoplasmic reticulum (ER) containing integral membrane proteins of the smooth and rough ER. Although lacking nuclear pore complexes and lamina, the R-rings derive specifically from the inner nuclear membrane. These findings are consistent with the idea that all transmembrane proteins synthesized in the ER and the outer nuclear membrane can freely diffuse through the pore membrane domain into the inner membrane. Uniquely, the soluble transfected Nopp140 is directly involved in the generation of these membrane stacks as it localizes to the electron dense matrix in which they are embedded. The only well-documented example of intranuclear membrane proliferation is the nucleolar channel system of the postovulation human endometrium. The transient emergence of the nucleolar channel system correlates precisely with the readiness of the endometrium for the implantation of the fertilized egg. The nucleolar channel system exhibits an ultrastructure that is indistinguishable from R-rings, and nuclei of human endometrium harbor Nopp140 and ER marker containing structures. Therefore, the nucleolar channel system appears to be identical to the R-rings, suggesting a role for Nopp140 in human reproduction.

The endometrial nucleolar channel system as an indicator of progestin potency in HRT

A quantitative assessment has been made of nucleolar channel systems (NCS) in the endometrial glands of postmenopausal women receiving hormone replacement therapy. The women were taking conjugated equine oestrogen and one of five progestins. The number of NCS induced was related to the dose of progestin administered. The minimum doses of progestin inducing a comparable response to premenopausal secretory phase endometria were found to be 1-2.5 mg norethindrone, 150 micrograms norgestrel and 20 mg dydrogesterone. Progesterone and medroxyprogesterone acetate were inadequate at the doses tested. The results show that the quantification of endometrial gland NCS would be a useful addition to the biochemical and morphological assessments made of any new progestin treatment.

Origin and fate of the nucleolar channel system of normal human endometrium

Human normal endometrium was examined in ultrathin sections. Nucleolar channel system (NCS) appeared in the endometrial epithelial cells during the early and mid secretory phase of menstrual cycle. The NCS was a hollow ball like structure of different sizes and was composed of 2 to 5 rows of tubules embedded in an amorphous matrix. On its surface there were numerous electron dense particles resembling ribosomes. It was usually located within or associated with the nucleolus. Sometimes, it was close to the nuclear envelope or protruding out from the nucleus. On occasion, NCS with simplified structure was found in the perinuclear cytoplasm. Concepts concerning the genesis, involution and function(s) of the NCS were discussed.

Human fetal endometrium--light and electron microscopic study.

Human fetal endometrium was examined by light and electron microscopy. Our study shows the following new morphological aspects: (1) Glands are already present. (2) Endometrium undergoes a maturation process during gestation and at late gestational age resembles late proliferative endometrium. (3) The nuclear bodies are present in cell nuclei throughout gestation. (4) Nucleolar channel systems (NCS) sometimes appear at a late gestational age. (5) Cells with the same morphology as that of endocrine cells are found in the basal layers of endometrium at a late gestational age. The significance of these morphological aspects is discussed.