User:Z3331264

Lab Attendance

Lab 1--Z3331264 11:49, 25 July 2012 (EST)

Lab 2--Z3331264 10:02, 1 August 2012 (EST)

Lab 3--Z3331264 10:02, 8 August 2012 (EST)

Lab 4--Z3331264 11:08, 15 August 2012 (EST)

Lab 5--Z3331264 10:33, 22 August 2012 (EST)

Lab 6--Z3331264 10:29, 29 August 2012 (EST)

Lab 7--Z3331264 10:12, 12 September 2012 (EST)

Lab 1: Fertilisation

2010 Nobel Prize Winner in Physiology or Medicine

Robert G. Edwards, For the development of in vitro fertilisation

For more Information

Recent Article on Fertilisation

Adiponectin and its receptors modulate granulosa cell and cumulus cell functions, fertility, and early embryo development in the mouse and human.

The expression of Adiponectin in mouse and human follicle cells was studied. Additionally, the function of this hormone in regulating fertilisation and early embryo development was observed. Adiponectin has been demonstrated to be secreted by adipocytes as well as ovarian cells. Their role in modulating metabolic homeostasis in granulosa and cumulus oophorus cells has also been studied. This study took into consideration, the impact of changing metabolic homeostasis on not only granulosa but also cumulus cells and thus the quality of the oocyte, pre-fertilisation. Adiponectin was shown to function as a cytokine and the levels of its receptors ADIPOR1 and ADIPOR2 were shown to be statistically significantly related to fertility outcome.

Consequently, adiponectin can enhance the quality of the oocyte pre-fertilisation as well as positively impact on embryonic development. While the particular genes involved in the response to adiponectin require further study, the applications of these results are promising. The addition of adiponectin to the maturation media of oocytes in human infertility care may improve the developmental competence of mature oocytes and enhance the possibility of successful in vitro fertilisation.

Interestingly, women with Polycystic Ovary Syndrome have lower levels of adiponectin which in turn alter the metabolic, steridogenic and apoptiotic activities of these cells. Such impacts have been hypothesised to be correlated with the lack of fertility in this cohort. Consequently, adjustments of adiponectin levels in treatment of this syndrome is a promising future research area.

Article on PubMed

<pubmed>22633650</pubmed>

Lab 2:Embryo Development

Protein required for Fertilisation

Implantation

The transcription factor CCAAT enhancer-binding protein β (C/EBPβ) plays a major role during decidualisation of the uterine stromal cells. Silencing of this protein suppressed the expression of Lamc1 which encodes for laminin. This protein is secreted by decidual cells as a constituent of the extracellular matrix (ECM). The loss of laminin impaired the ECM architecture and stromal cell differentiation. As a result of the impaired formation of a basal lamina-like matrix, trophoblast outgrowth is reduced and the progression of embryo implantation is prevented.

<pubmed>21471197</pubmed>

Lab 3

Gestational Age vs. post-fertilisation Age

The post-fertilization age is the age since fertilization of the egg while gestational age is age since the first day of the mother's last menstrual cycle before fertilisation has occurred. Gestational age is approximately two weeks greater than post-fertilization age. Gestational age is used clinically because its start date can be clearly determined from the mothers account and so is more accurate. On the other hand, the moment of fertilization must be inferred by adding 14 days, a variable time frame.

Fishton, P.M (2011) Embryo Fetus Development Stages [Internet]. Available from: http://www.livestrong.com/article/92683-embryo-fetus-development-stages/ [Last accessed 13/8/2012]

Tissue derived from somites

Initially the paraxial mesoderm undergoes segmentation to form the sclerotome and dermomyotome. Subsequent differentiation of the sclerotome results in the development of the vertebrae and Intervertebral discs. Additionally, the dermomyotome divides into the dermatome, which contributes to the dermis of the skin throughout the trunk and the myotome which forms the epaxial(dorsal) and hypaxial(ventrolateral) skeletal muscles of the body wall.

Dermis

The dermis is the connective tissue that supports the epidermis and binds it to the hypodermis. It consists of two indistinct layers, the superficial papillary layer and deeper reticular layer. The thin papillary layer is composed of loose connective tissue with populations of fibroblasts, mast cells, macrophages and often leucocytes that have been extravasated. This layer interdigitates with the epidermis, the external layer of skin separated from the dermis by a basement membrane. The reticular layer is a thicker layer composed of irregular dense connective tissue. In comparison with the papillary layer it has more fibers and fewer cells. The presence of elastic fibres allows for the elasticity of the skin. A rich supply of sympathetic effector nerves, hair follicles and gland structures are derived from the dermis.

Vertebrae

The vertebral column consists of a series of small bones. Each vertebra is lined by a thin outer layer of periosteum, a vascular fibrous layer surrounding bone, except over articular surfaces. It has an outer layer of collagen with elastic fibers. It provides vascular and nerve supply to bone. The medullary cavity of bone is lined with endosteum, a thin CT of osteoprogenitor cells and osteoblasts. The cortical region of vertbrae is composed of compact lamella. The unit of compact bone is the osteon, which are concentric layers of mineralised matrix surrounding a central vertical blood vessel and nerve carrying canal. This canal is lined by endosteum. Each osteon also has concentrically arranged osteocytes with radiating canaliculi allowing for communication with other osteocytes.Volkman's canals are horizontal canals which allow a connection between osteons. Spongy bone is an interconnected network of trabecular and many intertrabecular spaces which fill up the medullary cavity. The laminated structure is due to the arrangement of the collagen fibres within the trabeculae giving the bone its strength. The trabecular spaces are filled with bone marrow and is the site of hematopoiesis.

Muscle

Skeletal muscle consists of long, cylindrical multinucleated cells, forming muscle fibers. The oval nuclei are located at the periphery of the cell, just under the membrane. These multinucleated fibers create the endomysium, a delicate connective tissue to surround the fiber in conjunction with fibroblasts and reticular fibers. These individual fibers form fascicles that are surrounded by the perimysium, a thin septa of dense connective tissue extending inwards from the epimysium, which surrounds the collection of fascicles that make up the skeletal muscle. Blood vessels form a rich capillary network in the endomysium, while larger blood vessels and lymphatic vessels are found in the other layers. The epimysium is known to taper off and show continuity with the tendons. Motor nerves branch out within the perimysium connective tissue to give rise to several terminal nerves which may innervate a single muscle fibre or multiple at once (motor unit).

Mescher, L.A. (2010) Junqueira's Basic Histology. McGraw Hill, Singapore. Chapters 5,7,8.

Lab 4

Invasive Prenatal Diagnostics

Amniocentesis

This procedure is performed at a gestational age between 15 and 18 weeks. The amniotic fluid is sampled by inserting a needle through the mother's anterior abdominal and uterine walls to pierce the chorion and amnion. Approximately 15 to 20ml can be safely withdrawn. Real time ultrasonography is used as guidance for the physician by outlining the position of the fetus and placenta. Fetal cells can be separated from the amniotic fluid and karyotyped in order to detect for genetic abnormalities such as Trisomy 21 (Down Syndrome). Additionally, analysis of the alpha-fetoprotein levels can indicate neural-tube defects such as anencephaly and spina bifida.

Chorionic Villus Sampling

This procedure is performed at a much earlier gestational age of 10 weeks compared to Amniocentesis although has a 1% higher risk of miscarriage. Biopsies of 5-20mg of trophoblastic tissue are obtained by either a transabdominal needle insertion or transcervically, by passing a polyethylene catheter through the cervix guided by real-time ultrasonography. Chorionic Villus sampling tests for genetic abnormalities such as Trisomy 21, and X-linked disorders as well as inborn errors of metabolism.

Moore, K. L., Persaud, T. V. N. & Torchia, M. G. (2013). The Developing Human (9th ed.). Philadelphia, PA: Elsevier Saunders.

Cord Stem Cells Therapy

A study was conducted on mesenchymal cells with stem cell potential from Wharton's Jelly of the umbilical cord(HUMSCs). In this study, HUMSCs were isolated and transformed into dopaminergic neurons in vitro. These neuron-like cells were able to express neurofilament, functional mRNAs responsible for the syntheses of subunits of receptors capable of generating an inward current in response to neurotransmitters such as glutamate, an abnormality seen in patients with Parkinson's disease. These dopaminergic neurons were then transplanted into the striatum of rats that were previously made parkinsonian by the unilateral striatal lesioning with a neurotoxin(6-hydroxydopamine HCl).

The success rate of transplantation was characterised by positive staining for tyrosine hydroxylase (TH), the rate-limiting catecholaminergic synthesising enzyme, and the release of dopamine into the culture medium. The success rate of the transplantation was 12.7% and of these, the therapeutic outcome was indicated by a partially corrected lesion-induced amphetamine-evoked rotation. Rats with unilateral lesions to the substantia nigra rotate in response to amphetamine, and other dopaminergic receptor agonists where the number of rotations is directly proportional to the degree of denervation. Therefore, the cohort with the highest rotations benefited the least from therapy. The transplantation of invitro-differentiated HUMSCs alleviated the lesion-induced amphetamine-evoked rotation in the Parkinsonian rats, demonstrating potential therapeutic values. Additonally, a four month follow up after transplantation identified the prolonged viability of the transplanted cells and thus have the potential to treat human parkinson's patients.

The study's findings may have a significant impact on the study of Parkinson's disease and potentially help to circumvent worrying ethical issues. However before human studies, the success rate of transplantation must be improved as well as observation of the effects and side-effects for transplantations beyond 1 year. Such effects include behavioral effects, secretion of transmitters, activation of microglia, release of cytokines (such as tumor necrosis factor-α and interleukin-1β), and possible development of brain tumor. Finally, the toxicity of the growth factor (SHH and FGF8) and medium used should be examined.

<pubmed>16099997</pubmed>

Lab 7

Myosatellite cells

Myosatellite cells are mononuclear quiescent progenitor cells found sandwiched between the sarcolemma and basal lamina of a myofibre that become activated durin mechanical strain to augment existing or form new muscle fibres. <pubmed>PMC1571137</pubmed>

Satellite cell activation

Two instances where satellite cells are activated include muscle mechanical strain during exercise and muscle damage. During intense exercise, the forces generated by activation combined with stretch mean that the sarcomeres may be pulled out to such a degree that there is no longer any overlap of the actin and myosin filaments, thus causing damage. Following damage, it is believed that initial and pulsar release of mechanosensitive growth factor(MGF), results in activation of satellite cells. Alternatively, at the injured site, recruitment of inflammatory cells results, and the subsequent release of cytokines as well as Fibroblast Growth Factor (FGF) have been shown to activate myosatellite cells. Once satellite cells are activated, the release of cyclins allows the cells to come out of the G0 phase of growth, increase mRNA expression and so protein synthesis. This allows for microfiber replacement, regeneration or hypertrophy.

<pubmed>PMC1571137</pubmed>

Effects of long term motor nerve damage on Skeletal Muscle

After long term damage to motor nerves that innervate skeletal muscle, such as spinal cord injury(SCI),changes in fiber type and fiber size have been reported. Studies have shown that a progressive decrease in fiber diameter is observed with the extent of atrophy being directly proportional with the age of the injury. Studies have also shown that change in muscle fiber type to fast fibers accompanies muscle atrophy following SCI. A study of the paretic soleus muscle of a SCI patient cohort, that normally is predominantly composed of slow type 1 fibres, showed a shift to type 2b fibres 7-10 months post SCI. These changes have been observed as commencing four months after initial injury when there is a reported decrease of mitochondria, and build up lipid vacuoles within the fibre.The loss of mitochondria has been attributed to the immobilised, disused and so atrophic muscle of patients. The impairment of the mitochondrial oxidative enzyme activities accompanies morphological changes and also explains the build up of lipid vacuoles, the common energy source for mitochondria. Changes in to fast fibres has also been used to explain the fatigability encountered during muscle rehab exercises.

Scelsi R,2001, 'Skeletal Muscle Pathology after Spinal Cord Injury' Basic Appl Myol, 11(2):75-85.