User:Z3418779

Lab attendance

Lab 2 attended though didn't know we had to do attendance on our page

Lab 3 --Z3418779 --Z3418779 (talk) 11:36, 20 August 2014 (EST)

Lab 4 --Z3418779 --Z3418779 (talk) 11:39, 27 August 2014 (EST)

Lab 6 --Z3418779 (talk) 11:27, 10 September 2014 (EST)

Lab 7 --Z3418779 (talk) 11:56, 17 September 2014 (EST)

Lab 8 --Z3418779 (talk) 11:27, 24 September 2014 (EST)

Lab Task 1; Research Paper Summaries

Li M, Zhao H-C, Li R, Yu Y, Qiao J (2014) Chromosomal Aberrations in In-Vitro Matured Oocytes Influence Implantation and Ongoing Pregnancy Rates in a Mouse Model Undergoing Intracytoplasmic Sperm Injection. PLoS ONE 9(7): e103347. doi:10.1371/journal.pone.0103347

In-vitro fertilisation (IVF) and In-vitro maturation (IVM) are potential ways to alleviate issues with gonadotrophin stimulation. IVF has been accepted as the prefered Assisted Reproductive technique because of the higher success rate then IVM, which has 7 to 12% probablitly of plantation. Previous studies have concluded that the low success rate is related to the etiology of infertility rather then the technique itself. The selection of specific oocytes based on morphological features can greatly increase chance of implantation.

The study goal is to analyse spindle and chromosome and spindle configurations during the maturation process and resulting pre- and post-implantation embryos.

Experiment 1: used immature oocytes collected and matured in-vitro, with 3 cultures one at 18, 20 and 22 hours. alpha-tubulin and chromosome configurations were imaged by immunofluorescnce method. Experiment 2: used intracytoplasmic sperm injection to fertilise IVM oocytes, the pre-implanation phase was then compared to that of fertilised IVo oocytes. Expierement 3: Resulting embryos where introduced to a Pseudo-pregnant mouse, followed by dissection of fetuses on days 6.5 and 12.5.

Results; Spindle assembly in expiement 1 showed 18h had less that 50% originally, then rose to 52% in metaphase,the 20 and 22 hour grounds have significantly higher abnormalities (p<0.05) though had lower then a fresh control group. In expierment 2 1072 immature oocytes were collected and cultured with 924 becoming mature at MII stage, with 18h and 20h groups having reduced gene expression compared to 22h and control. Experiment 3 no significant difference in embryos on day 6.5, with 18h having slightly lower fetal rate then other groups. Day 12.5 showed a significantly reduced implantation rate in 18h group in comparison to control and 22 h groups. Full term development results average number of pups 18h=3, 20h=4.5, 22h =4.4, IVO=5.88

Shows greater chance of chromosome abnormalities in embryos from IVM timing and the relationship with post-implantation development in mouse models.

Shi W, Xu B, Wu L-M, Jin R-T, Luan H-B, et al. (2014) Oocytes with a Dark Zona Pellucida Demonstrate Lower Fertilization, Implantation and Clinical Pregnancy Rates in IVF/ICSI Cycles. PLoS ONE 9(2): e89409. doi:10.1371/journal.pone.0089409

Previous studies have shown abnormal morphology of oocytes and embryos results in lower viability for pre-implantation embryos and increase early pregnancy loss. This study focused on one specific oocyte abnormality a Dark Zona Pellucida (DZP)which previous studies have concluded to not significantly impact fertilization, embryo quality and pregnancy rate. Unlike previous studies DZP embryos would be seperated from NZP (Normal Zona Pellucida)

Patient population consisted of 268 infertile couples aged less than 38 years, being treated with IVRF or ICSI64 patients were put on embryo transfer cycles with occyte surronded by DZP being further subdivide based on percentage of Dark Zona pellucida; Group A(47) had 58% of Zona Pellucida dark on average and Group B(22) had entierly Dark Zona Pellucida. The remaining 204 patients made up the control group with NZP.

All patients were stimulated using a long pituitary down -regulation protocol. Oocytes were cultured in fertilisation medium. Sperm was used according to density gradient. Fertilized oocytes were placed in cleavage medium. Twenty mature oocytes (10 NZP, 10 DZP) were examined using JEOL-1230 Transmission Electron Microscope.

Reuslts; Miscarriages and live births for the three groups had no large differences. Though there were marked differences in fertilisation rate, frequency of high quality embryos, implantation rates and clinical pregnancy rates. All being lower Group B compared to control. Comparing the microscopic morphology shows no change in thickness of Zona Pellucida. NZP showed typical typical mitochondria shape; oval to spherical in shape with dense matrix and few cristae. DZP Mitochondria displayed swollen mitochondria encircling or containing vacuoles.

Study concluded that higher DZp results in decreased fertilisation rate, low rate of high quality embryos, adverse pregnancy outcomes and increased abnormal mitochondria and cytoplasmic vacuoles.

--Mark Hill These are relevant references and good summaries. You have not formatted the reference links correctly, see Help:Reference_Tutorial (4/5).

Lab Task 2; Image upload

Fetal Skeletal Muscle Progenitors [[1]]

Sakai H, Sato T, Sakurai H, Yamamoto T, Hanaoka K, et al. (2013) Fetal Skeletal Muscle Progenitors Have Regenerative Capacity after Intramuscular Engraftment in Dystrophin Deficient Mice. PLoS ONE 8(5): e63016. doi:10.1371/journal.pone.0063016

--Mark Hill Good image uploaded correctly. Reference link is not formatted. Help:Reference_Tutorial (4/5)

Lab Task 3

References

Julie R. Fuchs, Shinichi Terada, Didier Hannouche, Erin R. Ochoa, Joseph P. Vacanti, Dario O. Fauza.Engineered fetal cartilage: Structural and functional analysis in vitro. Journal of Pediatric Surgery Volume 37, Issue 12, Pages 1720–1725, December 2002

Sayer AA1, Cooper C.Fetal programming of body composition and musculoskeletal development.Early Hum Dev. 2005 Sep;81(9):735-44.

Baróti B1, Pap Z, Pánti Z, Buruian MM, Pávai Z. Morphometric and ultrasonographic study of the human fetal hip joint during intrauterine development. Rom J Morphol Embryol. 2013;54(4):977-81.

Liberty G1, Boldes R, Shen O, Shaul C, Cohen SM, Yagel S. The fetal larynx and pharynx: structure and development on two- and three-dimensional ultrasound. Ultrasound Obstet Gynecol. 2013 Aug;42(2):140-8. doi: 10.1002/uog.12358. Epub 2013 Jul 16.

--Mark Hill Three relevant references, link is not formatted correctly Help:Reference_Tutorial You should have included a sentence on why these articles were selected. (4/5)

Lab Task 4

Part 1

Identify a paper that uses cord stem cells therapeutically and write a brief (2-3 paragraph) description of the paper's findings.

Stem cell therapy and curcumin synergistically enhance recovery from spinal cord injury. [[2]]

Ormond DR, Shannon C, Oppenheim J, Zeman R, Das K, et al. (2014) Stem Cell Therapy and Curcumin Synergistically Enhance Recovery from Spinal Cord Injury. PLoS ONE 9(2): e88916. doi:10.1371/journal.pone.0088916

This paper investigates the result of Stem cell micro injection on spinal cord injury recovery and the effect curcumin(anti-inflammatory form turmeric) on Stem Cell Proliferation.

Findings; The addition of low concentrations of curcumin(500nM) results in a 180% increase in neurosphere proliferation,concentrations equal or higher then 1um fragements neurospheres and causes apotptosis. BBB Scores for Moderate SCI; Scores in week 5 and 6 showed a 33% higher score in treated rats compared to control. There was no significant difference between NSC and NSC/curcumin groups. BBB Scores for Severe SCI; After 2 weeks NSC and NSC/curcumin show similarly significant improvement compared to control, this improvement degrades in later weeks. Body weight all rats showed a decrease in weight then increase corresponding to recovery, except in Severe SCI in which curcumin alone showed improvement in week 5. Soleus Muscle Mass; in Moderate SCI showed no significant differentiation, in Severe SCI all treatemnt groups had higher mass then control particularly combination NSC/curcumin. Histopathological analysis;NSC and NSC/Curcumin displayed better recovery by comparing spared area to total area

Study displays Neural stem Cell therapy with curcumin is effective in recovery in Spinal cord injury. In the case of Moderate SCI effect of NSC and NSC/Curcumin showed a similarly significant recovery increase. In Severe SCI NSC/Curcumin had a greater aid in recovery. Curcumin assists recovery by reducing inflammation and gliosis, allowing unobstructed increased availability of neurotrophic factors from neural stem cells. The increased proliferation in-vitro supports the notion proliferative properties of curcumin.

Part 2

There are a number of developmental vascular "shunts" present in the embryo, that are closed postnatally. Identify these shunts and their anatomical location.

The Cardiovascular system has three important shunts present in the developing embryo, allowing blood to flow to differ from typical postnatal circulation. The key developmental shunts are Foramen Ovale,Ductus Venosus, Ductus Arteriosus.

Foramen Ovale; present in the Interatrial septum connecting the Left atrium and Right atrium. Allows oxygenated blood to bypass the lungs and enter the arterial network immediately. Usually open at birth, soon closed by two flaps of interatrial setum forming the fossa ovalis.

Ductus Venosus; connection of the umbilical vein and portal vein and inferior vena cava, purpose is to allow blood to bypass the liver. Functionally closes at birth and structurally closes within first week.

Ductus Arteriosus; connection of pulmonary artery and proximal ascending aorta, become ligamentum arteriosum after closure at birth

Lab Task 5

Anorectal Malformation

Anorectal Malformation is a defect of the anus and rectum which occurs during fetal development. There are multiple classifications of anorectal malformation based on the associated fistula. Low lesion;colon lies close to skin with an resulting in narrowing of the anus and rectum ending in blind pouch. High lesion; anus opens into bladder or genitalia, ,Persistent cloaca; rectum, vagina and urethra are joined into a single channel.

Causes; Abnormal development during weeks 7-10 as the lower section of intestinal tract forms. Originally the lower section large intestine and urinary tract are a single mass of cells. during the 7th-10th week this single mass of cells differentiates, seperate and form their appropriate organs. There is no correlation between activity of mother during pregnancy and incidence of malformation, more common in boys then girls.

Anorectal malformation occurs in 1 of 5,000 babies. Is associated with multiple other conditions and disorders these include; spinal , congenital, tracheal, esophageal, kidney, urinary and limb defects.

Lab Task 6

Identify and write a brief description of the findings of a recent research paper on development of one of the endocrine organs covered in today's practical.

Reciprocal epithelial: endothelial paracrine interactions during thyroid development govern follicular organization and C-cells differentiation

Study investigates how endothelial cells are recruited into the developing thyroid, and wether they control thyrocytes, C-cell differentiation and glandular organization. Thyroid explants were micro dissected from Vegfa-floxed and Pax8-cre mice embryos, then cultured in gelatin-coated dishes. Embryos were fixed and RNA probed for Vegfa and VEGF. Dissected thyroids were fixed in formaldehyde and some sections were immunofluorescence. Total RNA was extracted from embryonic thyroid lobes and underwent Real time polymerase chain reaction. Control and cKO lobes were rinsed and stained with uranyl acetate and examined under an electron microscope.

Results; Through out developmental stages of Endothelial cells closely surround thyroid epithelium; VEGFA knockout mice die around the period of childbirth, Reduction in vascular density hinders reorganization of epithelial mass into mono-layers because of polarization(shown by lack of basal pole). Additionally the thyroid developmental processes of bilobation, differentiation, polarization and vascularization are reproducible in a invivo culture, if adequate oxygen and nutrients are supplied

Findings; VEGFA is increased in a hypoxic environment via Hypoxia-induced factor-1 (HIF1A). When polarity normalizes decreases VEGFA expression. VEGFA derived from epithelium recruits the dense endothelial network in thyroid and promotes expansion. Endothelial cells are crucial for reorganization of follicular cells into follicles and differentiation of C-cells.

Continual interaction of epithelial and endothelial cells determines extent of thyroid development.

Hick, A., Delmarcelle, A., Bouquet, M., Klotz, S., Copetti, T., Forez, C., Van Der Smissen, P., Sonveaux, P., Collet, J., Feron, O. and others, (2013). Reciprocal epithelial: endothelial paracrine interactions during thyroid development govern follicular organization and C-cells differentiation. Developmental biology, 381(1), pp.227--240.

http://dev.biologists.org/content/early/2014/09/05/dev.110833.short

Identify the embryonic layers and tissues that contribute to the developing teeth.

Embryonic layers; Mesoderm, Ectoderm, neural crest ectomesenchyme all contribute to teeth development;

Ectoderm; contributes to Ameloblasts which later form tooth enamel

Neural crest mesenchyme; contributes to odontoblasts which undergoes dentinogenesis forming dentin

Mesoderm: forms blood supply of dental pulp

http://www.embryology.ch/anglais/sdigestive/gesicht05.html

Lab Task 8

Testis development

Indifferent development of gonads Originates from intermediate mesoderm forming genital ridges, pronephros appears with pronephric duct and nephrogenic mesenchyme, mesonephros appears with mesonephric tubule. Both later will fully or partially degenerate. Gonads descend down to pelvic region.

Gonadal differentiation Presence of Y chromsome has 48 protein-coding genes which begin the differentiation of the gonads into testes. In around week 6 Sertoli cells begin the appear in the primordia, to instruct germ cells; which have migrated through the primative streak. Leydig appear begin to secrete testosterone. The germ cells are enveloped in Sertoli cells which stop thier differentiation at T1 prospermatogonia until after birth.

In the testes the paramesoneph degenerates because of anti0Mullerian hormone secreted by the Sertoli cells. Mesonephric duct remains and under testosterone further differentiates to later form rete testis and ductus deferens. Testis cords of Sertoli and Germ Cells differentiate into semiferous tubules.

External genitalia differentiation Indifferentiation stage in which cloaca is divided by proliferating mesencyme forming the urorectal septum. The presence of Dihydrotestosterone result in growth of genital tubercle and formation of genital folds. The folds are maintained then fuse creating perineal and penile raphe. Lateral to urethral folds is the labioscrotal swelling which later becomes the scrotum.