ANSC 2400 3rd Edition Lecture 6 Outline of Last Lecture I. Ontogeny of the Reproductive System (Cont.) A. Primitive Gut and the Urogenital SystemB. Epithelial CellsC. Formation of the Broad LigamentII. Comparative Female Reproductive AnatomyD. Ruminants E. Bovine ModelF. Mare TractG. PigH. Humans (Primates)I. Bovine Tract In-SituOutline of Current Lecture I. Embryonic Quail Blood-Staining II. Primordial Germ Cells III. Comparative Aspects of Female AnatomyA. EweB. SowC. MareD. DogE. CatCurrent LectureI. Embryonic Quail Blood-Staining- Embryonic quail blood at the time of primordial germ cells circulation viewed with different interfaces opties. These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- Same microscope field viewed by Fluorescence Microscopy following immunocytochemical staining with SSEA1, a stem cell marker- Note: Larger PGC’s and smaller blood cells.- One of the early steps of this whole process is the differentiation of some of the early blastocyst cells, the epiblast cells to differentiate into what are called the primordial germ cells. - These will become the gametes in the adult gonad and we talked about some ways to identify them and characterize them.- Avian species – The migration of primordial germ cells is actually in the circulation. They enter the circulation at the site of formation and then they are deposited in thedeveloping gonadal ridge.- Noticeably larger cells.- Since they are being transported into the blood, it can be easy to draw a few microliters of blood from an early embryo at the right stage (which is about 4-5 days of incubation) and look at it under the microscope. - In avian – mostly nucleated cells.- Treat them with an anti-body to surface the molecule on the surface of the primordial germ cell. - (SSEA1) – Stem cell marker: Can see it on the surface on a lot of cells but not red blood cells. - If there are primordial germ cells present this antibody should bind to the surface marker/protein- Once it binds to the SSEA1 on the surface of the primordial germ cell, then we take a second antibody that is conjugated to a fluorescent dye of some sort. - There is a whole range of these in many different colors. - Conjugate the fluorescent dye to the secondary antibody to the first antibody (Image: Like a sandwich) and then the second antibody with the fluorescent dyes only where the primary antibody to SSEA1 is located and therefore identifies that cellas varying SSEA1 surface marker.- Look at under fluorescence only the cells that are stained with fluorescent dyes showup the two larger cells are also the cells that are specific for SSEA1 based on our double antibody technique that we are using.- If we take the primary antibody to SSEA1 and do the same thing, but with the secondantibody instead of conjugating a fluorescent dye to that we make it magnetic then we can literally put the sample in a test tube and put the test tube in a magnetic fieldand that magnet will draw the bound cells over to the margins of the tube and all theother cells will just go running through. We can get rid of all those cells and remove the tube from the magnet and wash the cells of interest out.- Called Magnetic Activated Cell Sorting (MACS) – Tremendous procedure. - Cant purifier them but you can enrich/concentrate them.II. Primordial Germ Cells - True Stem cells derived from the epiblast- Bearing stem cell markers. - Can use that to advantage to verify what those cells are and to even enrich them in a preparation. III. Comparative Aspects of Female Anatomy - Figure 2-2- Female tract is going to be closely associated with the pelvic girdle.- The female tract supported by the broad ligament bi-laterally. - Everything starts out as bi-lateral - Bi-lateral ovaries - Bi-lateral paramesonephric ducts, which then become female organs - Extension of the body wall bi-laterally grows into the abdominal cavity and it extendsthe supported structures medially and then those will join at varying degrees and either remain bi-lateral or fuse to form a single structure. - Figure 2-5- Figure 2-13A. Ewe- Is another Ruminant, so it has common features that other ruminants have.- The Ewe is like a smaller version of the bovine.~ (The U-Tract cut Open – The ventral floor of the entire tract)- The vestibule of the vagina rises from the urogenital sinus.Differences in the Ewe - Ovary - Breeds of sheep tend to ovulate more than one oocyte.- Ewes will ovulate two, three or sometimes more oocytes depending upon the breed of sheep.- Multiple ovulations- Multiplets- Multiple Graafian Follicles and all can ovulate- Post Ovulation – can see multiple CLsOviduct- Embryonated edge of the infundibulum, or funnel shaped portion.- Proximal to the ovary- Ampulla and the Isthmus distal to the infundibulum.- Supported to the Mesosalpinx ligament forms the ovarian bursa.Caruncles - Figure 2-20- Also, common in other Ruminants.- Roughly around a hundred or so.- Attached to the fetal placenta to provide faster exchange between the fetus and the dam.- Occasionally, in the pigmented breeds sometimes the caruncles will have pigmentation in some of the reproductive structures. - One of the places the pigment will often reside is in the caruncles. Annular Rings (Also, called the cervical rings)- See more cervical rings in the ewe than in the bovine. - Annular Rings – Folds that close off the cervix.- Figure 2-3 (Cow)- Figure 2-7 (Sow)A. Sow- Polytocous - (litter-bearers)Ovary- Multiple ovulations from both sides- Figure 2-13 (Sow)- Figure 2-17 (Sow)(In-situ)- The uterine horns are lying down in the abdominal cavity.- The uterine horns seem to be compartmentalized, because they contain a conceptus. A pregnant tract. The weight of the contents of the uterine horn helps to sink it down into the abdominal cavity. Pregnant tract will be deeper (interior edge) front of the pelvic floor. - Each ovulation that occurs results in an individual conceptus (fetus plus its own embryonic membranes) Ex. Placenta- Multiple structures in a post pubertal animal that suggests multiple follicles are forming or are about to ovulate/have ovulates and CLs will form in their place.- The CL’s will form individually from each ovulation point; each follicle that will begin to differentiate into luteal cells but eventually a lot of the luteal cells will almost in-mesh with the rest of the follicles are they