Phase 1 safety, tolerability, pharmacokinetics and pharmacodynamics results of a long-acting C-type natriuretic peptide prodrug, TransCon CNP.

AIM: TransCon CNP is a novel prodrug designed to provide sustained release of C-type natriuretic peptide (CNP) for once-weekly therapy, addressing the pathology leading to aberrant skeletal development in achondroplasia. This phase 1 trial was initiated to assess the safety, tolerability, pharmacodynamics (PD) and pharmacokinetics (PK) of TransCon CNP. METHODS: This randomized, placebo-controlled, single-ascending dose phase 1 trial was performed at two sites in Australia and enrolled 45 healthy adult males. Subjects received placebo or TransCon CNP (single-ascending dose cohorts [3, 10, 25, 75 or 150 µg CNP/kg]). The primary endpoint was frequency of adverse events and other safety outcomes. Other endpoints included PK and PD measured by cyclic guanosine-monophosphate (cGMP) and amino-terminal propeptide of CNP (NTproCNP). RESULTS: TransCon CNP provided continuous systemic exposure to CNP over at least 7 days post-dose. Plasma and urine levels of cGMP were significantly increased in subjects administered TransCon CNP at 75-150 µg CNP/kg, indicating target engagement of active CNP at the natriuretic peptide receptor-B (NPR-B) for at least 1 week post-dose. TransCon CNP was well-tolerated, with no serious treatment-emergent adverse events or discontinuations. Extensive cardiac safety assessments did not reveal any clinically relevant effects on electrocardiogram parameters, including heart rate, PR, QRS and QTcF intervals. CONCLUSIONS: Safety and PD data from this phase 1 trial support that TransCon CNP is well tolerated, with a PK profile compatible with a once-weekly dosing regimen. Further studies are ongoing to evaluate the potential of TransCon CNP to positively impact abnormal endochondral ossification in children with achondroplasia.

Functioning and well-being in older children and adolescents with achondroplasia: A qualitative study.

The study aimed to explore how having achondroplasia affects older children and adolescents' day-to-day functioning and well-being. Individual/focus group interviews were conducted with older children/adolescents between the ages of 9 to <18 years and diagnosed with achondroplasia to elicit key concepts. An adapted grounded theory approach informed the qualitative analysis of interview data. Thirty-two children and adolescents completed interviews. Study results revealed five impact domains, including physical health, functioning, school impacts, emotional well-being, and social well-being. Frequently reported impacts on physical health included low stamina/tiring easily (81%) and back pain (69%). Key impacts in the functioning domain were difficulty with reaching objects or high places (84%) and walking long distances (75%). Emotional impacts included feeling different (63%), worried/scared (47%), and embarrassed/self-conscious (47%). Impacts on social well-being included difficulty with sports or physical play (81%) and others treating child as younger than their actual age (75%). The most frequent school impact was trouble participating in physical education (81%). A preliminary theoretical model depicting the experiences of older children/adolescents with achondroplasia was constructed based on the analysis. The preliminary theoretical model of older children and adolescents' experiences of living with achondroplasia may be used to inform future research and clinical practice.

A qualitative study of the impacts of having an infant or young child with achondroplasia on parent well-being.

BACKGROUND: Currently, there is limited research on how having a child diagnosed with achondroplasia affects parents' lives. The purpose of the study was to investigate the experiences of parents of infants and young children less than two years of age with achondroplasia. METHODS: Concept elicitation interviews were conducted with parents of children less than 2 years of age with achondroplasia in the United States and Spain. Using grounded theory methods modified for health outcomes research, a qualitative analysis of interview transcripts was conducted. Based on the qualitative analysis, a preliminary theoretical model of the experiences of parents of infants and young children with achondroplasia was developed. RESULTS: Fifteen parents, including 14 mothers and 1 father from 15 unique families, participated in individual or focus group concept elicitation interviews in the US (n = 9) and Spain (n = 6). The qualitative analysis identified four key parent impact domains, which included caretaking responsibilities, impacts on emotional well-being, having worries and concerns about their child, and impacts on daily well-being. Frequently discussed caretaking responsibilities among parents were managing child's medical care/treatment (93%), obtaining adaptations/items for child (73%), and monitoring child to avoid complications of achondroplasia (67%). Emotional impacts included feeling stressed/overwhelmed (67%), depressed/sad (40%), and anxious/nervous (33%). Worries and concerns included worry/concern about the future (100%), concerns regarding child's physical health (87%), worry about child's social well-being (80%), concern for child's emotional well-being (73%), and worry about child being able to function independently (67%). Daily well-being impacts included family strain (60%), missed work time (47%), and missed/limited social activities (33%). Based on the qualitative findings, a preliminary theoretical model depicting the experiences of parents of infants and young children with achondroplasia was created. CONCLUSIONS: The study sheds light on the range of impacts that parents of infants and young children with achondroplasia may experience, including caretaking responsibilities, impacts on emotional well-being, worries/concerns regarding their child, and impacts on daily well-being. The theoretical model of parent experiences may provide a helpful framework for informing future research and clinical practice.

Assessing physical symptoms, daily functioning, and well-being in children with achondroplasia.

This study's purpose was to provide qualitative evidence to support the development of two observer-reported outcome measures assessing the physical symptoms/complications of achondroplasia in children and impacts on children's quality of life. Individual/focus group concept elicitation interviews were conducted with parents of children aged 2 to <12 years with achondroplasia and experts. Qualitative analysis of transcripts, based on an adapted grounded theory approach, informed item generation and measure development. Cognitive debriefing (CD) interviews were conducted with parents to confirm relevance and understanding. Thirty-six parents participated in concept elicitation interviews. The analysis identified major physical symptoms/complications and impacts of achondroplasia, which informed the development of the Achondroplasia Child Experience Measures (ACEMs): ACEM-Symptom and ACEM-Impact. ACEM-Symptom was comprised of eight major symptoms/complications including pain (58%), ear infections/fluid in ear (56%), and low stamina/tiring easily (56%). ACEM-Impact consisted of 31 major impacts in the domains of daily functioning, emotional well-being, social well-being, and need for assistance/adaptive devices. Impacts on functioning included difficulty reaching objects/high places (89%) and toileting (67%). Emotional impacts included feeling different (53%) and feeling frustrated/annoyed (47%). Social impacts included difficulty participating in sports/physical play (86%) and being treated as younger than age (83%). Following CD interviews with 16 additional parents, validation-ready ACEM measures were generated. The study improves our understanding of the experiences of children with achondroplasia and provides evidence supporting the content validity of the ACEMs. Validated ACEMs may be used to assess potential benefits of future treatments for comorbidities of achondroplasia.

Assessing the impacts of having a child with achondroplasia on parent well-being.

PURPOSE: This study's purpose was to develop a better understanding of the experiences of parents of children with achondroplasia and to provide qualitative evidence to support the development of a patient-reported outcome (PRO) measure of parent impacts. METHODS: Concept elicitation (CE) individual/focus group interviews were conducted with parents of children aged 2 to < 12 years with achondroplasia in the United States and Spain. The qualitative analysis informed the PRO measure development. Cognitive debriefing (CD) interviews were conducted to ensure parent understanding and item relevance. RESULTS: Thirty-six parents participated in individual/focus group CE interviews. The analysis identified parent impacts in four domains, including caretaking responsibilities, emotional well-being, family, and work, and results informed the development of the Achondroplasia Parent Experience Measure (APEM). Caretaking responsibilities included managing child's medical care (92%), helping child with self-care (67%), advocating for child (64%), assisting child (56%), and observing/monitoring child (e.g., to ensure safety; 47%). Impacts on parents' emotional well-being included worry about the future (75%), worry about child's physical health (67%), safety concerns (50%), feeling stressed/overwhelmed (44%), and worry about child's social relationships (42%). Impacts on family and work included family strain (56%), limiting/adapting family activities (42%), and missed work time (50%). CD interviews with an additional 16 parents of children with achondroplasia confirmed understanding and item relevance. CONCLUSION: The results improve our understanding of the experiences of parents of children with achondroplasia and provide qualitative evidence to support the content validity of the APEM. A psychometric study is needed to validate the measure.

Direct demonstration of a neonatal Fc receptor (FcRn)-driven endosomal sorting pathway for cellular recycling of albumin.

Albumin is the most abundant plasma protein involved in the transport of many compounds, such as fatty acids, bilirubin, and heme. The endothelial cellular neonatal Fc receptor (FcRn) has been suggested to play a central role in maintaining high albumin plasma levels through a cellular recycling pathway. However, direct mapping of this process is still lacking. This work presents the use of wild-type and engineered recombinant albumins with either decreased or increased FcRn affinity in combination with a low or high FcRn-expressing endothelium cell line to clearly define the FcRn involvement, intracellular pathway, and kinetics of albumin trafficking by flow cytometry, quantitative confocal microscopy, and an albumin-recycling assay. We found that cellular albumin internalization was proportional to FcRn expression and albumin-binding affinity. Albumin accumulation in early endosomes was independent of FcRn-binding affinity, but differences in FcRn-binding affinities significantly affected the albumin distribution between late endosomes and lysosomes. Unlike albumin with low FcRn-binding affinity, albumin with high FcRn-binding affinity was directed less to the lysosomes, suggestive of FcRn-directed albumin salvage from lysosomal degradation. Furthermore, the amount of recycled albumin in cell culture media corresponded to FcRn-binding affinity, with a ∼3.3-fold increase after 1 h for the high FcRn-binding albumin variant compared with wild-type albumin. Together, these findings uncover an FcRn-dependent endosomal cellular-sorting pathway that has great importance in describing fundamental mechanisms of intracellular albumin recycling and the possibility to tune albumin-based therapeutic effects by FcRn-binding affinity.

Generation of a double transgenic humanized neonatal Fc receptor (FcRn)/albumin mouse to study the pharmacokinetics of albumin-linked drugs.

Human serum albumin (HSA) is a natural carrier protein possessing multiple ligand binding sites with a plasma half-life ~19days, facilitated by interaction with the human neonatal Fc receptor (FcRn), that promotes it as a highly attractive drug delivery technology. A lack of adequate rodent models, however, is a major challenge in the preclinical development of albumin-linked therapeutics. This work describes the first double transgenic mouse model bearing both human FcRn and HSA genes (hFcRn(+/+), hAlb(+/+)) under the control of an endogenous promoter. Human FcRn was shown by immunohistochemical and qPCR analysis to be ubiquitously expressed in the major organs. Physiological levels of HSA were detected in the blood that exhibited similar FcRn binding kinetics to recombinant or human serum-derived HSA. The circulatory half-life (t1/2) was shown to be dependent on FcRn binding affinity that increased from low affinity (t1/2 29h), to wild type (t1/2 50h), to high affinity (t1/2 80h) variants, that validates the application of the model for optimizing the pharmacokinetics of drug carriers who's circulatory half-life is dependent in some manner upon interaction with endogenous FcRn. This study presents a novel mouse model that better mimics the human physiological conditions and, thus, has potential wide applications in the development of albumin-linked drugs or conventional drugs whose action is influenced by reversible binding to endogenous HSA.

Extending serum half-life of albumin by engineering neonatal Fc receptor (FcRn) binding.

A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals.

Optimizing thrombelastography (TEG) assay conditions to monitor rFVIIa (NovoSeven) therapy in haemophilia A patients.

INTRODUCTION: There is no established laboratory method that can predict the most optimal dose of bypassing agents for treatment of haemophilia A. The objectives of the study was to develop an assay that can a) differentiate between the haemostatic capacity in blood from healthy individuals and severe and moderate haemophilia patients; b) show a dose-response correlation to rFVIIa addition; and c) show dose response differences of rFVIIa addition to plasma samples from non-inhibitor patients of different severity. MATERIALS AND METHODS: Citrated whole blood from 25 haemophilia A patients was used in four thrombelastography (TEG) assays initiated with: 1) kaolin, 2) Tissue Factor (TF, Innovin 1:42,500), 3) TF 1:42,500+1.2 nM tPA (tissue plasminogen activator) or 4) TF 1:200,000. rFVIIa was added to give a final concentration in the range of 0.02-4.8 microg/ml. RESULTS: The TEG assays showed large differences in clot formation demonstrated by prolonged clotting time (R-time), decreased maximum thrombus generation (MTG) between severe and moderate haemophilia A patients and between haemophilia patients and healthy males. The maximal amplitudes (MA) of the clot and resistance against fibrinolysis were only compromised when TF with tPA was added. CONCLUSION: In vitro addition of rFVIIa improved all TEG profiles significantly in a dose-dependent manner; but only the TEG assay containing kaolin could differentiate between the rFVIIa doses, showing that blood from severe patients need higher doses of rFVIIa to normalize the clot formation profile compared to blood from moderate patients. Kaolin seems to be the most useful TEG assay for monitoring rFVIIa treatment.

rFVIIa and NN1731 reduce bleeding in hydroxyethyl starch hemodiluted rabbits.

BACKGROUND: Colloid plasma expanders are used to maintain blood pressure and ensure tissue perfusion during hypovolemia, e.g., caused by traumatic bleeding. Although colloids stabilize the cardiovascular system, they can also potentially cause coagulopathy. Consequently, bleeding tendency may increase, as well as the associated risk of morbidity and mortality. Thus, there is a need for hemostatic treatment options for these patients. rFVIIa (NovoSeven, Novo Nordisk A/S, Bagsvaerd, Denmark) is a hemostatic agent that effectively controls bleedings in patients with inhibitor-complicated hemophilia. rFVIIa works by enhancing thrombin generation on the activated platelet surface at the site of injury, leading to the formation of a stable fibrin clot. NN1731 is an rFVIIa analog with increased hemostatic potential and is currently under clinical development. METHODS: In this study, the effect of rFVIIa and NN1731 on cuticle bleeding in rabbits 50% hemodiluted with hydroxyethyl starch (molecular weight ∼ 200,000) was tested. Cuticle bleeding was induced after a two-stage hemodilution procedure. After 5 minutes, the animals were treated with rFVIIa (2, 5, or 10 mg/kg), NN1731 (1 or 2 mg/kg), or vehicle, followed by 30 minutes of observation. RESULTS: Hemodilution caused a significant increase in bleeding time and blood loss. rFVIIa dose-dependently reduced bleeding time and blood loss, reaching statistical significance at 10 mg/kg. However, 2 mg/kg NN1731 reduced bleeding time and blood loss significantly and to a similar extent as 10 mg/kg rFVIIa. This increased hemostatic potential of NN1731 compared with rFVIIa and was confirmed by findings using thromboelastography on ex vivo hemodiluted whole blood. CONCLUSION: In summary, rFVIIa and NN1731 significantly and dose-dependently reduced bleeding in extensively hemodiluted rabbits.

Differential clot stabilising effects of rFVIIa and rFXIII-A2 in whole blood from thrombocytopenic patients and healthy volunteers.

The haemostatic effect of recombinant activated factor VII (rFVIIa;NovoSeven) in thrombocytopenic patients has been a matter of controversy. Haemostasis by rFVIIa occurs via FVIIa-mediated thrombin generation in a platelet-dependent manner and may therefore be suboptimal in patients without functional platelets. Under such conditions, a clot-stabilizing agent, such as factor XIII (FXIII), may supplement the effect ofrFVIIa and improve haemostasis. Recombinant factor XIII (rFXIII-A2) is produced as an A2 homodimer of the FXIII A subunit and is equivalent to cellular FXIII normally found in platelets. The combined effects of rFVIIa andrFXIII-A2 were evaluated in clot lysis assays using factor XIII-deficient plasma and by whole blood thrombelastography (TEG) analysis from normal donors and thrombocytopenic stem cell transplantation patients. Clotting time was shortened by rFVIIa (0.6-10 microg/ml). rFVIIa only modestly improved anti-fibrinolysis,whereas rFXIII-A2 (0-20 microg/ml) enhanced anti-fibrinolysis without effect on clotting time. TEG analysis showed rFVIIa shortened the clotting time, and enhanced clot development, maximal mechanical strength and resistance to fibrinolysis, whereas, rFXIII-A2 enhanced clot development,maximal mechanical strength and markedly enhanced resistance to fibrinolysis. These data illustrate that rFVIIa and rFXIII-A2 contribute to clot formation and stability by different mechanisms suggesting enhanced haemostatic efficacy by combining these agents.

Investigation of the effect of kaolin and tissue-factor-activated citrated whole blood, on clot-forming variables, as evaluated by thromboelastography.

BACKGROUND: The Thrombelastograph (TEG; Haemoscope Corp.) analyzes clot formation in whole blood (WB) and treatment based on this analysis has been shown to reduce transfusion requirements in liver and cardiac surgery when compared to conventional coagulation analysis. Implementing TEG as a routine laboratory-based analysis, however, requires validation of the activators employed and the effect of storage of the WB sample in citrate before analysis. STUDY DESIGN AND METHODS: The effect of kaolin, tissue factor (TF) 1:17,000, or TF 1:42,500 on TEG clotting time (R), Angle (velocity of clot formation), and maximum clot strength (amplitude [MA]) were evaluated, together with day-to-day variation, the coefficient of variance (CV%), and the effect of citrate storage time. RESULTS: Clot formation variables were equally affected by TF 1:17,000 and kaolin activation, whereas R was significantly longer when TF 1:42,500 was used. The CV for the different variables varied from 3 to 13 percent with no significant differences between assays. Storage of citrated WB significantly affected the TEG variables in a hypercoagulable direction. Only the R, however, was significantly affected (12%) when samples rested for 0 and 30 minutes were evaluated with kaolin as the activator. CONCLUSION: The TEG assays evaluated were reproducible and present with an acceptable CV% for routine clinical practice. Kaolin and TF 1:17,000 equally affected the clot formation variables. Storage of WB for up to 30 minutes in citrate did not, except for R, affect clot formation variables when kaolin was used as activator allowing for immediate analysis when the sample arrives in the laboratory.

Evaluation of thromboelastography for monitoring recombinant activated factor VII ex vivo in haemophilia A and B patients with inhibitors: a multicentre trial.

Predicting the clinical effect of bypassing agents such as recombinant activated factor VII in haemophilia patients with inhibitors is hampered by the limited availability of reliable laboratory monitoring tools. This multicentre, open-label trial aimed to explore the dose-response relationship between recombinant activated factor VII concentration and thromboelastography parameters in blood samples from patients with haemophilia A or B with inhibitors in a nonbleeding state. Citrated whole blood samples from 16 patients (>or=16 years) with haemophilia A or B were spiked ex vivo with recombinant activated factor VII (1.2, 1.6, 2.0, 2.6, 3.0, 3.5 microg/ml), corresponding approximately to doses of 90-270 microg/kg. Samples were analysed by Thromboelastograph or Rotation Thromboelastography (three United States and three European centres, respectively) within 30 min (final lipidated recombinant tissue factor 1: 17 000; final CaCl2 15 mM). Thromboelastograph/Rotation Thromboelastography parameters showed large intersubject variation in the baseline profiles. There was a clear effect when recombinant activated factor VII was added; however, a clear concentration-response relationship was only detected for one patient. This is likely due to the fact that the curves were not sufficiently abnormal that led to reduced assay sensitivity. Our preliminary results suggest that thromboelastography may potentially be a clinically useful tool for monitoring changing concentrations of recombinant activated factor VII in haemophilia patients, but only when the baseline curve is significantly abnormal. Thus, test conditions may need to be optimized before Thromboelastograph/Rotation Thromboelastography can be utilized for all inhibitor patients.

Benzo[b]thiophene-2-carboxamides and benzo[b]furan-2-carboxamides are potent antagonists of the human H3-receptor.

Benzo[b]thiophene-2-carboxamides and benzo[b]furan-2-carboxamides have been found to be antagonists on the human histamine-3-receptor, showing a Ki value of as low as 4 nM.

Cinnamic amides of (S)-2-(aminomethyl)pyrrolidines are potent H3 antagonists.

New imidazole-free H3 antagonists have been found in a series of cinnamic amides of (S)-(aminomethyl)pyrrolidines. The influence of the substituent on the aromatic moiety on the potency and the inhibition of three cytochrome P450 subtypes are also described.

Numerical chromosome errors in day 7 somatic nuclear transfer bovine blastocysts.

Day 7 bovine somatic nuclear transfer (NT) embryos reconstructed from granulosa cells were examined for numerical chromosome aberrations as a potential cause of the high embryonic and fetal loss observed in such embryos after transfer. The NT embryos were reconstructed using a zona-free manipulation method: half-cytoplasts were made from zona-free oocytes by bisection, after which two half-oocytes and one granulosa cell (serum-starved primary culture) were fused together and activated. The NT embryos were cultured in modified synthetic oviductal fluid containing essential and nonessential amino acids, myoinositol, sodium citrate, and 5% cattle serum in microwells for 7 days, at which time nuclei from all blastocysts were extracted and chromosome aberrations were evaluated using dual-color fluorescent in situ hybridization with bovine chromosome 6- and 7-specific probes. Five embryo clone families, consisting of 112 blastocysts reconstructed from five different primary granulosa cell cultures, were examined. Overall, the mean chromosome complement within embryos was 86.9 +/- 3.7% (mean +/- SEM) diploid, 2.6 +/- 0.5% triploid, 10.0 +/- 3.1% tetraploid, and 0.5 +/- 0.2% pentaploid or greater; the vast majority (>75%) of the abnormal nuclei were tetraploid. Completely diploid and mixoploid embryos represented 22.1 +/- 4.5% and 73.7 +/- 5.5%, respectively, of all clones. Six totally polyploid blastocysts, containing or=5N chromosome complements, respectively) between two clone families were different (P < 0.01), as were blastocyst yields between other clone families (P < 0.01). Blastocyst yield was not correlated to % total ploidy error between clone families, but an inverse relationship (P < 0.01) between blastocyst total cell number and total % chromosome abnormality was observed within embryos. Categorization of the blastocysts into three quality grades (good, medium, and poor) and comparison of the distribution of ploidies when classified into 0%, 0.1-5.0%, 5.1-10.0%, 10.1-15.0%, and 15.1-100% errors within embryos indicated that medium- and poor-grade embryos were different (P < 0.05) from good-quality, in vitro-produced embryos. In a separate study, 11 different granulosa cell cultures (that did not correspond to those used for NT) were evaluated and found to possess only 0.23 +/- 0.12% ploidy errors. These results demonstrate that 1) the percentage of ploidy errors in bovine NT blastocysts is inversely related to total blastocyst cell number, 2) the mixoploid condition is representative of the majority of embryos, 3) 100% polyploid NT blastocysts can exist, and 4) the ploidy errors seem not to be derived from the donor cells.

Bovine embryos contain a higher proportion of polyploid cells in the trophectoderm than in the embryonic disc.

The frequency of polyploid cells in the embryonic disc (ED) and in the trophectoderm (TE) was assessed in 50 in vitro produced bovine embryos fixed at days 7-8 post insemination (pi) and in 20 in vitro produced embryos that were transferred to uteri of recipients at day 7 and then recovered and fixed at day 12 pi. Separation of TE and ED cells was obtained by microdissection and the frequency of polyploid cells was determined by interphase cytogenetic analysis using fluorescence in situ hybridization (FISH) with chromosome 6- and chromosome 7-specific probes. The results show that 96% of day 7 embryos contain polyploid cells in the TE, whereas only 58% contain polyploid cells in the ED. In day 12 embryos 85% of TE and 40% of ED preparations contain polyploid cells. Statistical analysis revealed that the frequency of polyploid cells was significantly higher in the TE than in the ED in embryos containing less than 25% polyploid cells (n = 65). The few embryos (n = 5), which contained more than 25% polyploid cells, did not show this difference. Further, it was revealed that the level of polyploidy on day 7-8 was significantly higher than on day 12, both in the TE (two-fold) and in the ED (seven-fold).

Activation of the ribosomal RNA genes late in the third cell cycle of porcine embryos.

In porcine embryos, nucleoli are first observed during the third postfertilization cell cycle, i.e., at the 4-cell stage. However, direct studies of the initiation of rRNA transcription have not been reported. This transcription was investigated in the present study by simultaneous visualization of the rRNA genes and the rRNA by fluorescent in situ hybridization using a porcine 28S rDNA probe and subsequent visualization of argyrophilic nucleolar proteins by silver staining of extracted and fixed nuclei from in vivo-derived porcine embryos (n = 229). Nucleologenesis was observed by transmission electron microscopy. In general, the 2-cell and 4-cell embryos fixed at 10 and 20 h postcleavage (hpc) showed no signs of rRNA transcription. Four small clusters of fluorescein isothiocyanate (FITC) labeling were visible in interphase nuclei, consistent with hybridization to the rRNA gene clusters only; there was no silver staining at the sites of the rRNA genes and nucleolus precursor bodies. From 30 hpc onwards, most 4-cell embryos had medium size to large clusters of FITC-labeled areas colocalized with silver staining of rRNA gene clusters and fibrillogranular nucleoli. These observations indicate that rRNA transcription had been initiated. These signs of rRNA synthesis could be blocked by actinomycin D, which is a strong inhibitor of RNA polymerase I. The rRNA transcription of porcine embryos is initiated between 20 and 30 hpc, corresponding to the end of the S-phase or the beginning of the G2 phase during the third cell cycle.