Time course and pathologies of in vitro maturation and fertilization of bovine oocytes

In this study, bovine oocyte maturation and early fertilization stages were systematically investigated by confocal laser scanning microscopy (CLSM) and subsequent 3-D-reconstruction. Grade I and II oocytes were isolated from 2–8 mm follicles from slaughterhouse ovaries and fixed at different time points of in vitro maturation (IVM) and in vitro fertilization (IVF). After staining the DNA, Serine 10 phosphorylated histone H3 (H3S10p), microtubules and f-actin microfilaments with four different fluorescence dyes, the oocytes were imaged in toto by serial optical sections. In the subsequent analysis, meiotic stages from the germinal vesicle to the metaphase II were morphologically characterized, and the approximate time course was determined. Using the same methodological approach, the first stages of fertilization were investigated: sperm penetration into the oocyte, completion of oocyte meiosis, the development of the maternal and paternal pronucleus, the formation of the sperm aster and the approximate time course. In parallel, the anomalies detected during in vitro oocyte maturation and fertilization were characterized and classified to subsequently determine their incidence. This study provided a large collection of three-dimensional microscopic images of bovine oocytes at different stages of presumably normal maturation and fertilization. Moreover, a spectrum of different severe anomalies was documented. In total, 1078 oocytes were fixed at 2-hour intervals from 0 to 28 hours of IVM and analyzed. Nearly all oocytes fixed before IVM were still in the germinal vesicle (GV) stage, at 10 hours virtually all oocytes had resumed meiosis and the GV had been broken down. The large number of three-dimensional snapshots of oocytes was used to reconstruct the presumably normal formation of the spindle apparatus and the chromosome alignment in meiosis I and II. Moreover, a spectrum of anomalies of meiosis I was documented: from the formation of a multipolar spindle, incorrect positioning of the spindle to chromosome segregation errors and the occurrence of chromatin bridges in anaphase I. To investigate the early steps of fertilization, in total 654 oocytes were fixed at 4, 5, 6, 7, 8, 10 and 12 hours post insemination (hpi), imaged in toto and morphologically analyzed. Thereby, we determined the time frame of sperm VII. Summary 107 penetration and oocyte activation, and the approximate time course of the development of the maternal and paternal pronucleus and the sperm aster. The formation of the two pronuclei and of the sperm aster was divided into 5 stages. Thus, we could show how the sperm nucleus shortly after penetrating into the oocyte decondenses and transiently recondenses to a small dense paternal pronucleus which decondenses again. 24.9 % of the oocytes were arrested in meiosis before metaphase II or had been already spontaneously activated and could not be fertilized. 26.9% of the penetrated and activated oocytes had severe anomalies: 11.9 % were polyspermic, 17% showed meiotic aberrations of the oocyte. The results of this study contribute to further elucidate the complex processes of oocyte maturation and fertilization in cattle and other mammals including humans and to improve the diagnostics and therapies of fertility problems.Zeitlicher Ablauf und Fehler bei der in vitro Reifung und Befruchtung boviner Eizellen In dieser Arbeit wurden systematisch die Reifung und die ersten Stadien der Befruchtung von Rinder-Eizellen mittels confocaler Laser-Scanning-Mikroskopie (CLSM) und nachfolgender 3-D-Rekonstruktion untersucht. Die Eizellen wurden aus Ovarien vom Schlachthof aus 2-8 mm Follikeln isoliert und zu unterschiedlichen Zeitpunkten der In-vitro-Maturation (IVM) und In-vitro Fertilisation (IVF) fixiert. Nach Anfärbung der DNA, Serin-10-phosphoryliertem Histon H3 (H3S10p), der Mikrotubuli und F-Actin-Filamente mit vier verschiedenen Fluoreszenzfarbstoffen wurden die Eizellen im Ganzen in optischen Serienschnitten aufgenommen. In der Auswertung wurden Stadien der Meiose vom Germinalvesikel bis zur Metaphase II morphologisch charakterisiert und der ungefähre Zeitablauf bestimmt. Mit dem gleichen methodischen Ansatz wurden die ersten Schritte der Befruchtung untersucht: das Eindringen des Spermiums und die Aktivierung der Eizelle, die Bildung des maternalen und paternalen Vorkerns, die Ausbildung des „Sperm Aster“ und der ungefähre Zeitablauf dieser Vorgänge. Parallel dazu wurden die bei der Eizellreifung und Befruchtung in vitro entdeckten Anomalien charakterisiert und klassifiziert, um anschließend ihre Häufigkeit zu bestimmen. Im Rahmen dieser Untersuchung entstand eine große Sammlung dreidimensionaler mikroskopischer Bilder, die Rinder-Eizellen in unterschiedlichen Stadien der mutmaßlich normalen Reifung und Befruchtung sowie ein Spektrum ganz unterschiedlicher schwerwiegender Anomalien erfasst. Insgesamt wurden 1078 Eizellen in 2-Stundenintervallen von 0 bis 28 Stunden IVM fixiert und analysiert. Nach unseren Beobachtungen waren nahezu alle Eizellen vor der in vitro Maturation im Germinalvesikel (GV) Stadium, nach 10 h hatten praktisch alle Eizellen die Meiose wieder aufgenommen und den GV aufgelöst. Aus der großen Zahl dreidimensionaler Momentaufnahmen wurden die mutmaßlich normale Ausbildung des Spindelapparates und die Anordnung der Chromosomen in der Meiose I und II rekonstruiert. Gleichzeitig wurde ein Spektrum von Anomalien der Meiose I dokumentiert, von der Bildung einer VI. Zusammenfassung 105 multipolaren Spindel, der falschen Positionierung der Spindel bis zu Fehlern bei der Chromosomensegregation und dem Auftreten von Chromatinbrücken in der Anaphase I. Zur Untersuchung des Befruchtungsvorgangs wurden insgesamt 654 Eizellen zu den Zeitpunkten 4, 5, 6, 7, 8, 10 und 12 Stunden nach der Besamung (hpi) im Ganzen aufgenommen und analysiert. Bei der Auswertung wurden der Zeitrahmen des Eindringens der Spermien und der Zeitablauf der Bildung des väterlichen und mütterlichen Vorkerns sowie des „Sperm Aster“ bestimmt. Die Entwicklung der beiden Vorkerne und des „Sperm Aster“ wurde jeweils in fünf Phasen unterteilt. So konnte dargestellt werden, wie der Kern des Spermiums kurz nach dem Eindringen dekondensiert, um vorübergehend wieder zu rekondensieren und einen zunächst kleinen dichten paternalen Vorkern zu bilden, der erneut expandiert. 24,9 % der Eizellen waren in der Meiose vor der Metaphase II arretiert oder waren bereits spontan aktiviert, und konnten so nicht mehr befruchtet werden. Bei 26,9 % der penetrierten und aktivierten Eizellen wurden schwerwiegende Anomalien gefunden: 11,9 % wiesen eine Polyspermie auf, 17 % Aberrationen der Meiose der Eizelle. Diese Ergebnisse dieser Untersuchungen tragen dazu bei, die komplexen Vorgänge der Eizellreifung und Befruchtung beim Rind und bei anderen Säugern einschließlich des Menschen weiter aufzuklären und die Diagnostik und Therapie von Fertilitätsstörungen zu verbessern

Time course and pathologies of in vitro maturation and fertilization of bovine oocytes

In this study, bovine oocyte maturation and early fertilization stages were systematically investigated by confocal laser scanning microscopy (CLSM) and subsequent 3-D-reconstruction. Grade I and II oocytes were isolated from 2–8 mm follicles from slaughterhouse ovaries and fixed at different time points of in vitro maturation (IVM) and in vitro fertilization (IVF). After staining the DNA, Serine 10 phosphorylated histone H3 (H3S10p), microtubules and f-actin microfilaments with four different fluorescence dyes, the oocytes were imaged in toto by serial optical sections. In the subsequent analysis, meiotic stages from the germinal vesicle to the metaphase II were morphologically characterized, and the approximate time course was determined. Using the same methodological approach, the first stages of fertilization were investigated: sperm penetration into the oocyte, completion of oocyte meiosis, the development of the maternal and paternal pronucleus, the formation of the sperm aster and the approximate time course. In parallel, the anomalies detected during in vitro oocyte maturation and fertilization were characterized and classified to subsequently determine their incidence. This study provided a large collection of three-dimensional microscopic images of bovine oocytes at different stages of presumably normal maturation and fertilization. Moreover, a spectrum of different severe anomalies was documented. In total, 1078 oocytes were fixed at 2-hour intervals from 0 to 28 hours of IVM and analyzed. Nearly all oocytes fixed before IVM were still in the germinal vesicle (GV) stage, at 10 hours virtually all oocytes had resumed meiosis and the GV had been broken down. The large number of three-dimensional snapshots of oocytes was used to reconstruct the presumably normal formation of the spindle apparatus and the chromosome alignment in meiosis I and II. Moreover, a spectrum of anomalies of meiosis I was documented: from the formation of a multipolar spindle, incorrect positioning of the spindle to chromosome segregation errors and the occurrence of chromatin bridges in anaphase I. To investigate the early steps of fertilization, in total 654 oocytes were fixed at 4, 5, 6, 7, 8, 10 and 12 hours post insemination (hpi), imaged in toto and morphologically analyzed. Thereby, we determined the time frame of sperm VII. Summary 107 penetration and oocyte activation, and the approximate time course of the development of the maternal and paternal pronucleus and the sperm aster. The formation of the two pronuclei and of the sperm aster was divided into 5 stages. Thus, we could show how the sperm nucleus shortly after penetrating into the oocyte decondenses and transiently recondenses to a small dense paternal pronucleus which decondenses again. 24.9 % of the oocytes were arrested in meiosis before metaphase II or had been already spontaneously activated and could not be fertilized. 26.9% of the penetrated and activated oocytes had severe anomalies: 11.9 % were polyspermic, 17% showed meiotic aberrations of the oocyte. The results of this study contribute to further elucidate the complex processes of oocyte maturation and fertilization in cattle and other mammals including humans and to improve the diagnostics and therapies of fertility problems.Zeitlicher Ablauf und Fehler bei der in vitro Reifung und Befruchtung boviner Eizellen In dieser Arbeit wurden systematisch die Reifung und die ersten Stadien der Befruchtung von Rinder-Eizellen mittels confocaler Laser-Scanning-Mikroskopie (CLSM) und nachfolgender 3-D-Rekonstruktion untersucht. Die Eizellen wurden aus Ovarien vom Schlachthof aus 2-8 mm Follikeln isoliert und zu unterschiedlichen Zeitpunkten der In-vitro-Maturation (IVM) und In-vitro Fertilisation (IVF) fixiert. Nach Anfärbung der DNA, Serin-10-phosphoryliertem Histon H3 (H3S10p), der Mikrotubuli und F-Actin-Filamente mit vier verschiedenen Fluoreszenzfarbstoffen wurden die Eizellen im Ganzen in optischen Serienschnitten aufgenommen. In der Auswertung wurden Stadien der Meiose vom Germinalvesikel bis zur Metaphase II morphologisch charakterisiert und der ungefähre Zeitablauf bestimmt. Mit dem gleichen methodischen Ansatz wurden die ersten Schritte der Befruchtung untersucht: das Eindringen des Spermiums und die Aktivierung der Eizelle, die Bildung des maternalen und paternalen Vorkerns, die Ausbildung des „Sperm Aster“ und der ungefähre Zeitablauf dieser Vorgänge. Parallel dazu wurden die bei der Eizellreifung und Befruchtung in vitro entdeckten Anomalien charakterisiert und klassifiziert, um anschließend ihre Häufigkeit zu bestimmen. Im Rahmen dieser Untersuchung entstand eine große Sammlung dreidimensionaler mikroskopischer Bilder, die Rinder-Eizellen in unterschiedlichen Stadien der mutmaßlich normalen Reifung und Befruchtung sowie ein Spektrum ganz unterschiedlicher schwerwiegender Anomalien erfasst. Insgesamt wurden 1078 Eizellen in 2-Stundenintervallen von 0 bis 28 Stunden IVM fixiert und analysiert. Nach unseren Beobachtungen waren nahezu alle Eizellen vor der in vitro Maturation im Germinalvesikel (GV) Stadium, nach 10 h hatten praktisch alle Eizellen die Meiose wieder aufgenommen und den GV aufgelöst. Aus der großen Zahl dreidimensionaler Momentaufnahmen wurden die mutmaßlich normale Ausbildung des Spindelapparates und die Anordnung der Chromosomen in der Meiose I und II rekonstruiert. Gleichzeitig wurde ein Spektrum von Anomalien der Meiose I dokumentiert, von der Bildung einer VI. Zusammenfassung 105 multipolaren Spindel, der falschen Positionierung der Spindel bis zu Fehlern bei der Chromosomensegregation und dem Auftreten von Chromatinbrücken in der Anaphase I. Zur Untersuchung des Befruchtungsvorgangs wurden insgesamt 654 Eizellen zu den Zeitpunkten 4, 5, 6, 7, 8, 10 und 12 Stunden nach der Besamung (hpi) im Ganzen aufgenommen und analysiert. Bei der Auswertung wurden der Zeitrahmen des Eindringens der Spermien und der Zeitablauf der Bildung des väterlichen und mütterlichen Vorkerns sowie des „Sperm Aster“ bestimmt. Die Entwicklung der beiden Vorkerne und des „Sperm Aster“ wurde jeweils in fünf Phasen unterteilt. So konnte dargestellt werden, wie der Kern des Spermiums kurz nach dem Eindringen dekondensiert, um vorübergehend wieder zu rekondensieren und einen zunächst kleinen dichten paternalen Vorkern zu bilden, der erneut expandiert. 24,9 % der Eizellen waren in der Meiose vor der Metaphase II arretiert oder waren bereits spontan aktiviert, und konnten so nicht mehr befruchtet werden. Bei 26,9 % der penetrierten und aktivierten Eizellen wurden schwerwiegende Anomalien gefunden: 11,9 % wiesen eine Polyspermie auf, 17 % Aberrationen der Meiose der Eizelle. Diese Ergebnisse dieser Untersuchungen tragen dazu bei, die komplexen Vorgänge der Eizellreifung und Befruchtung beim Rind und bei anderen Säugern einschließlich des Menschen weiter aufzuklären und die Diagnostik und Therapie von Fertilitätsstörungen zu verbessern

Colorectal Cancer Stage at Diagnosis Before vs During the COVID-19 Pandemic in Italy

IMPORTANCE Delays in screening programs and the reluctance of patients to seek medical attention because of the outbreak of SARS-CoV-2 could be associated with the risk of more advanced colorectal cancers at diagnosis. OBJECTIVE To evaluate whether the SARS-CoV-2 pandemic was associated with more advanced oncologic stage and change in clinical presentation for patients with colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS This retrospective, multicenter cohort study included all 17 938 adult patients who underwent surgery for colorectal cancer from March 1, 2020, to December 31, 2021 (pandemic period), and from January 1, 2018, to February 29, 2020 (prepandemic period), in 81 participating centers in Italy, including tertiary centers and community hospitals. Follow-up was 30 days from surgery. EXPOSURES Any type of surgical procedure for colorectal cancer, including explorative surgery, palliative procedures, and atypical or segmental resections. MAIN OUTCOMES AND MEASURES The primary outcome was advanced stage of colorectal cancer at diagnosis. Secondary outcomes were distant metastasis, T4 stage, aggressive biology (defined as cancer with at least 1 of the following characteristics: signet ring cells, mucinous tumor, budding, lymphovascular invasion, perineural invasion, and lymphangitis), stenotic lesion, emergency surgery, and palliative surgery. The independent association between the pandemic period and the outcomes was assessed using multivariate random-effects logistic regression, with hospital as the cluster variable. RESULTS A total of 17 938 patients (10 007 men [55.8%]; mean [SD] age, 70.6 [12.2] years) underwent surgery for colorectal cancer: 7796 (43.5%) during the pandemic period and 10 142 (56.5%) during the prepandemic period. Logistic regression indicated that the pandemic period was significantly associated with an increased rate of advanced-stage colorectal cancer (odds ratio [OR], 1.07; 95%CI, 1.01-1.13; P = .03), aggressive biology (OR, 1.32; 95%CI, 1.15-1.53; P < .001), and stenotic lesions (OR, 1.15; 95%CI, 1.01-1.31; P = .03). CONCLUSIONS AND RELEVANCE This cohort study suggests a significant association between the SARS-CoV-2 pandemic and the risk of a more advanced oncologic stage at diagnosis among patients undergoing surgery for colorectal cancer and might indicate a potential reduction of survival for these patients

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Evaluation of the systematic recording of diagnostic data in the Valdostana cattle

At present, in Italy no systematic recording of diagnostic data for improving animal health and welfare at farm level is available. A first approach towards a health recording system for cattle has been attempted in the Aosta Valley, recording the diagnoses of local Valdostana cattle between 2017 and 2018. The objectives of the present study were: (1) to evaluate the strengths and the critical points of the recording system and (2) to determine the incidence of specific diseases for the year 2018 in Valdostana cows. A standardised key with 69 specific diseases was used by 21 veterinarians for registering the diagnoses in a database. Data were collected from almost 80% of the farms present in the Aosta Valley. The main recorded diseases were those affecting the udder, reproductive apparatus, locomotor system and parasitic infections. Diseases affecting the respiratory and digestive system played a minor role. Since the general data loss through data validation was limited (8.8%), the recording system might be considered as an effective tool for gaining an objective overview of the farm health status. Nevertheless, some diagnoses in the recording system have to be more specified for allowing more precise epidemiologic insights.HIGHLIGHTS A health recording system enables farmers and veterinarians to improve animal health and welfare on farm level. Valdostana cattle show lower incidences for some health disorders when compared with literature data from other dairy cattle breeds. More specific diagnoses for parasitoses and claw disorders could be useful for breeding purpose

The Munich MIDY Pig Biobank - A unique resource for studying organ crosstalk in diabetes

OBJECTIVE: The prevalence of diabetes mellitus and associated complications is steadily increasing. As a resource for studying systemic consequences of chronic insulin insufficiency and hyperglycemia, we established a comprehensive biobank of long-term diabetic INSC94Y transgenic pigs, a model of mutant INS gene-induced diabetes of youth (MIDY), and of wild-type (WT) littermates. METHODS: Female MIDY pigs (n = 4) were maintained with suboptimal insulin treatment for 2 years, together with female WT littermates (n = 5). Plasma insulin, C-peptide and glucagon levels were regularly determined using specific immunoassays. In addition, clinical chemical, targeted metabolomics, and lipidomics analyses were performed. At age 2 years, all pigs were euthanized, necropsied, and a broad spectrum of tissues was taken by systematic uniform random sampling procedures. Total beta cell volume was determined by stereological methods. A pilot proteome analysis of pancreas, liver, and kidney cortex was performed by label free proteomics. RESULTS: MIDY pigs had elevated fasting plasma glucose and fructosamine concentrations, C-peptide levels that decreased with age and were undetectable at 2 years, and an 82% reduced total beta cell volume compared to WT. Plasma glucagon and beta hydroxybutyrate levels of MIDY pigs were chronically elevated, reflecting hallmarks of poorly controlled diabetes in humans. In total, ∼1900 samples of different body fluids (blood, serum, plasma, urine, cerebrospinal fluid, and synovial fluid) as well as ∼17,000 samples from ∼50 different tissues and organs were preserved to facilitate a plethora of morphological and molecular analyses. Principal component analyses of plasma targeted metabolomics and lipidomics data and of proteome profiles from pancreas, liver, and kidney cortex clearly separated MIDY and WT samples. CONCLUSIONS: The broad spectrum of well-defined biosamples in the Munich MIDY Pig Biobank that will be available to the scientific community provides a unique resource for systematic studies of organ crosstalk in diabetes in a multi-organ, multi-omics dimension