User:Z3333865

Lab Attendance

Lab 1 --Z3333865 11:00, 25 July 2012 (EST)

Lab 2 --Z3333865 10:04, 1 August 2012 (EST)

Lab 3 --Z3333865 11:58, 8 August 2012 (EST)

Lab Exercises

Lab 1

Question 1

As stated by IVF-worldwide, the history of In Vitro Fertilization (IVF) and embryo transfer (ET) dates back as early as the 1890s. Walter Heape, a professor and physician at the University of Cambridge, England, had been conducting research on reproduction in a number of animal species. He reported the first known case of embryo transplantation in rabbits, long before the applications to human fertility were even suggested. IVF history

IVF-worldwide also explains that in 1965, Robert Edwards together with Georgeanna and Howard Jones at Johns Hopkins Hospital in the USA attempted to fertilize human oocytes in vitro. The first IVF pregnancy was reported in 1973 by the Monash research team of Professors Carl Wood and John Leeton in Melbourne, Australia. Unfortunately, this resulted in early miscarriage. The first ever IVF birth occurred in Oldham, England on July 25, 1978. This birth was the result of the collaborative work of Patrick Steptoe and Robert Edwards. IVF history

Robert Edwards was awarded the 2010 Nobel Prize in Physiology or Medicine for the development of human In Vitro Fertilization (IVF) therapy. His achievements have made it possible to help treat infertility, which affects more than 1 in 10 couples worldwide. 2010 Nobel Prize

Question 2

<pubmed>PMC3353509</pubmed>

Traditional IVF methods involve the assisted fertilization of the oocytes with the spermatozoa. This is performed in the laboratory, whereby the physiological conditions to which the gametes are normally exposed in vivo are simulated. However, INVO (intravaginal culture of oocytes), is a simplified procedure and alternative option to conventional IVF. This assisted reproduction procedure uses the maternal vaginal cavity for incubation, instead of laboratory equipment.

Investigated in this study is the outcome of the INVO procedure and how this compares to the conventional IVF methods. Data was obtained regarding pregnancy, live birth, and single live birth rates. Results of this study showed that the INVO procedure had comparable successful rates with traditional IVF.

Statistics from 2008 on traditional IVF: the pregnancy, live birth, and singleton live birth rates per oocyte retrieval were 41.6%, 33.8%, and 23%, respectively.

Statistics from this study on INVO: the pregnancy, live birth, and singleton live birth rates per oocyte retrieval were 40%, 31.2%, and 24%, respectively.

The study also concluded that the most significant factor determining the success rate was the age of the mother. In terms of pregnancy, live birth, and single live birth rates, a significant decrease was observed across the groups of age from ≤29 until ≥40 years old. Results obtained by this study suggest that INVO procedures could be a viable alternative treatment for infertile patients.

Link to paper on INVO

Lab 2

In-class exercise

Confirmation of homologous recombination and c-MYC2 expression in ES cell clones.

Confirmation of homologous recombination and c-MYC2 expression in ES cell clones.

(A) Genomic DNA of ES cell clones 1, 14, 18 and 19 and of wildtype ES cells (wt Bruce 4) was digested with EcoRI. Digested DNA was analyzed by Southern blotting with a 5′ probe and a 3′ probe. (wt) DNA fragment of the wildtype c-Myc locus; (rec.) DNA-fragment of recombined hc-Myc locus. (B) Protein extracts were prepared of ES cell clones 1, 14, 18 and 19 as well as of wildtype ES cells (wt Bruce 4) and of a human lymphoblastoid cell line (LCL 1.11). Human c-MYC2 (hu. c-MYC, ca. 62 kDa) was detected with antibody clone Y69. In wildtype ES cells murine c-MYC2 (mu. c-MYC, ca. 64 kDa) was detected. For loading control an antibody specific for glyceraldehyde-3-phosphat-dehydrogenase (GAPDH; ca. 36 kDa) was used. Western blot results were reproduced five times.

Lehmann FM, Feicht S, Helm F, Maurberger A, Ladinig C, et al. (2012) Humanized c-Myc Mouse. PLoS ONE 7(7): e42021. doi:10.1371/journal.pone.0042021

Copyright: © 2012 Lehmann et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Assessment task

Jam2 expression in mouse uterus during early pregnancy.

Question 1

Jam2 expression in mouse uterus during early pregnancy.

(A) In situ hybridization of Jam2 mRNA. (B) Real-time RT-PCR quantification of Jam2 mRNA. (C) JAM2 immunostaining. D1, day 1; D2, day 2; D3, day 3; D4, day 4; D4.5-I, implantation site at day 4 midnight; D4.5-NI, inter-implantation site at day 4 midnight; D5-I, implantation site on day 5; D5-NI, inter-implantation site on day 5; PD3, day 3 of pseudopregnancy; PD4, day 4 of pseudopregnancy; Arrow, embryo. Bar = 150 µm.

Su R-W, Jia B, Ni H, Lei W, Yue S-L, et al. (2012) Junctional Adhesion Molecule 2 Mediates the Interaction between Hatched Blastocyst and Luminal Epithelium: Induction by Progesterone and LIF. PLoS ONE 7(4): e34325. doi:10.1371/journal.pone.0034325

Copyright: © 2012 Su et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Question 2

A protein associated with the implantation process is Hand2.

It is known that levels of this protein increase in uterine cells as progesterone levels rise. In a more recent NIH funded study, researchers discovered that Hand2 is also the switch that turns off estrogen’s stimulating effect on the epithelium.

For the study, the researchers developed a laboratory strain of mice in which the uterus fails to make Hand2. It was found that exposure to progesterone halted growth of the uterine epithelium in mice with functioning genes for Hand2. However, despite exposure to progesterone, epithelial growth continued unchecked in the mice without Hand2 genes.

Furthermore, at the time of implantation, Hand2 was expressed in uterine cells that lie beneath the surface layer of epithelial cells. Experiments have shown that estrogen stimulates the production of growth factors, which cause cells in the epithelial layer to multiply and grow. When progesterone is produced, it spurs the release of Hand2, which stops the production of growth factors. The uterine epithelial cells then stop multiplying, mature, and become receptive to the embryo. This is a key step in the process of implantation.

<pubmed>PMC3320855</pubmed>

Lab 3

Question 1

The post-fertilization age is the time that has passed since fertilization of the egg. The gestational age, however, is measured from the first day of the woman's last menstrual cycle to the current date. A normal pregnancy can range from 38 to 42 weeks.

The gestational age is approximately two weeks greater than post-fertilization age. Gestational age is more clinically significant because its start date can be clearly determined both before and after birth, whereas the exact moment of fertilization must be inferred.

Question 2

The somites developed from paraxial mesoderm, and will give rise to sclerotome, dermatome and myotome tissues.

The sclerotome relates to the axial skeleton and the proper functioning of the vertebral column: Sonic hedgehog signalling causes the ventromedial portion of the somite to differentiate into sclerotome.The sclerotome then develops into cartilage (chondrocytes) due to the transcription factor Pax 1.

Dorsolaterally, the dermomyotome develops first, which then differentiates into the dorsal dermatome and the ventral myotome.

The dermatome will contribute to the formation of the dermis due to the neurotrophin 3 factor. The dermis consists of: firstly, a more superficial papillary layer which has fine collagen and elastic fibres and contains small blood vessels (arterioles and capillaries), lymph and nerves. Secondly, a deeper reticular layer with dense collagen fibres and thick elastic fibres and it contains lymph, vascular plexus, nerves and appendages.

The ventral myotome can be split up into the epaxial myotome (dorsomedial quarter) and the hypaxial myotome (dorsolateral quarter). The epaxial myotome will result in formation of the erector spinae muscles and the hypaxial myotome will give rise to muscles of the trunk (ventrally) and limbs. The type of muscle which is formed is skeletal muscle - striated, multinucleated myofibers. Proteins such as Wnt 1 and 3 are related to the expression of genes which will cause muscle development.

Online Embryology course developed by the universities of Fribourg, Lausanne and Bern with the support of the Swiss Virtual Campus - Somite development.