1. Hyperactivation is a swimming pattern observed in mammalian sperm in the oviduct that is essential for fertilization. This swimming pattern is a circling movement caused by increased asymmetry of flagellar beating. The vigorous movement of hyperactivated sperm enables them to move more efficiently in the mucus and cumulus matrix surrounding the oocyte in the oviduct. There is a strong correlation between sperm hyperactivated motility and the ability to fertilize. Normal sperm motility Hyperactivated sperm motility

2. Note: Flagellar bend A laboratory exercise was developed to enable students to examine the effects of caffeinated drinks on sperm motility. The vigorous movement of hyperactivated sperm enables them to move more efficiently in the mucus and cumulus matrix surrounding the oocyte in the oviduct. Caffeine has been demonstrated to induce hyperactivation by raising intracellular Ca2. Teachers can purchase bull semen frozen in egg yolk extender and packaged in 0.5 ml plastic artificial insemination straws from West Hill Supply (Ithaca, NY). Working in pairs, students thaw semen by submerging the straw in a 39C water bath for 30 sec. The straw is then emptied into 2 ml of Carnation Instant Non-Fat Milk prepared with distilled water as per instructions on the package, and brought to a pH of 8.3 using 1 molar NaOH. The students can then observe the sperm under a microscope to see their linear progressive swimming movements. Students will then add coffee (adjusted to pH 8.3 with 1 M NaOH) to sperm at a 2:5 dilution and observe them immediately. Approximately 50% of the sperm are killed by the introduction of coffee; however, those that survive exhibit hyperactivation. As a control, students will treat sperm with decaffeinated coffee. As with the caffeinated coffee, 50% will not survive; however, of those that do, none will show hyperactivated movements. Development of the classroom activity was supported by a “Research Experiences for Teachers” supplement to NSF grant MCB- 0421855.

3. Oogeneis is the formation of eggs in the ovaries. Eggs develop from immature cells call oogonia, which are usually surrounded by a follicle. In humans the primary oocytes are in prophase of the first meiotic division by the time they are born. When the female reaches sexual maturity, once a month these primary oocytes finish meiosis and develop into a functional egg. Increases in estrogen levels cause a thickening of the uterine lining. Leutinizing hormone (LH) levels increase in response to the increase in estrogen. This determines ovulation. Following ovulation, the corpus luteum produces progesterone which prepares and maintains the uterus for the fertilized egg. The egg is unable to move, contains a nucleus and often a stored food in the form of yolk. The egg is usually larger than the sperm It is essential for the gametes (ova from the female and sperm from the male) to meet for fertilization to occur. Most animals exhibit an estrous cycle which is a limited period of sexual receptivity. Humans do not experience changes in sexual receptivity, however, they do experience changes in the uterine lining during what is called a menstrual cycle.

4. Spermatogesis is the production of sperm in the testes. Sperm develop from immature sex cells call spermatognia.. In humans, after a male sexually matures, some spermatogonia continue to divide mitotically while others develop into functional sperm. Most sperm are microscopic, containing a head, middle piece, and a flagellum. The head is made up of a nucleus, which contains the chromosomes and an acrosome which contains enzymes that help the sperm penetrate the egg. The middle piece contains mitochondria, which give the sperm energy to move. The flagellum enables the sperm to swim. A diploid zygote is formed from the joining of the haploid sperm cell with the haploid egg cell. When a sperm comes in contact with an egg, the acrosome releases enzymes that dissolve an opening through the protective membranes of the egg. Once a sperm has penetrated the egg, the formation of a fertilization membrane around the egg prevents other sperm from penetrating.

5. Purpose: Hyperactivation is a circling movement of mammalian sperm that is necessary for fertilization. It has been observed that Ca 2+ is necessary for hyperactivated motility. It is unclear as to the mechanism that triggers the movement of Ca 2+ into the flagellum to initiate hyperactivation. However, when caffeine was introduced to bull sperm hyperactivation was immediately induced. When bull sperm are introduced to caffeinated products, such as Pepsi, coffee and tea, the same effect can be observed. Working in pairs, students thaw semen by submerging the straw in a 39C water bath for 30 sec. The straw is then emptied into 2 ml of Carnation Instant Non-Fat Milk prepared with distilled water as per instructions on the package, and brought to a pH of 8.3 using 1 molar NaOH. The students can then observe the sperm under a microscope to see their linear, progressive swimming movements. Students will then add coffee (adjusted to pH 8.3 with 1 M NaOH to sperm at a 2:5 dilution and observe them immediately.

6. 2. Sperm are then diluted with a solution of Carnation Instant Non-fat milk. 1. Frozen bull sperm are thawed by submerging them in a 35 o water bath for 30 seconds. 3. Caffeinated products are then added to bull sperm and the effects are observed under the microscope. 4. As controls, decaffeinated versions of each product are added to sperm. At Genex cooperative in Ithaca, New York bull semen are collected using a long tube heated to 120 degrees F, affixed with a ring of warm water at the base. After collection, the semen is than diluted with whole milk, tested, prepared and placed into insemination straws. 1. 2. 3.

7. Materials: Petri dish 1 ml pipettes Indicator paper Distilled water 1 molar NaOH Bull sperm* Thermometer Hot water bath Folger’s Instant Coffee Decaffeinated coffee Lipton tea Decaffeinated tea Decaffeinated Pepsi Pepsi Beakers *West Hill Catalog number: RD501 (Phone: 607-330-0963)

8. Step 1. Step 2. Step 3/4. Lab Prep: Step 1: Frozen semen should be placed in a hot water bath at 35-37C. for 35 seconds immediately upon opening the shipment. Step 2: Prepare milk adding 9.58 grams of Carnation Instant Nonfat Milk to distilled water up to a total volume of 100 ml. Step 3: Use 1 molar NaOH, a couple of drops at a time, to bring the pH of the milk up to 8.3 Step 4: Heat 30 ml of the above milk for 1 minute (not to boiling), and add a Lipton Tea bag for 5-6 minutes until tea- like in appearance (light brown color).

9. Step 5: Prepare another 30 ml of milk as above, except use Lipton decaffeinated tea for a control. Step 6: Add a couple of drops at a time of 1 molar NaOH to both the caffeinated and uncaffeinated tea., until the pH is 8.3. Step 7: Heat 30 ml of the above milk (as specified for the tea) and add ¼ of a teaspoon of Folger’s coffee. Step 8: Prepare another 30 ml of milk as above, except use Folgers decaffeinated coffee for a control. Step 9: Add a couple of drops at a time of 1 molar NaOH to both the caffeinated and uncaffeinated coffee until the pH is 8.3. Step 10: Take 30 ml of Pepsi that has been left opened for an hour to de-carbonate, and add small amounts of NaOH, until the pH is 8.3. Step 9. Step 5.

10. Step 1: Place 1.0 ml of Milk into a test tube. Add the plastic straw of bull semen at 35C to this same test tube. Mix gently by tapping the side of the test tube with your finger. Step 2: Place 0.25 ml of bull semen / milk mixture (using a plastic pipette) onto a microscope slide. Add a cover slip. Step 3: Observe the bull semen using the 40X objective on your microscope (note: with the 40X objective and 10X eyepiece, the magnification is 400X). Step 4: Note observations on chart 1. Step 5: Place 0.125 ml of bull semen / milk mixture on a slide. Using a fresh pipette add.008 ml of coffee onto this same slide (you should just have to touch the bull semen mixture with the drop of coffee hanging from the pipette). Step 6: Note observations on chart 1 Step 7: Repeat steps 5 and 6 using the tea. Step 8: Note observations on chart 1 Step 9: Repeat steps 5 and 6 using Pepsi. Step 10: Note observations on chart 1 Observations Bull Sperm/milk mixture Mixture and coffee Mixture & decaf. coffee Mixture & tea Mixture & decaf. Tea Mixture & Pepsi Mixture & decaf. Pepsi

11. The scientific method- A universal approach to problem that allows other scientists to repeat the work or test its conclusions. Defining the problem – A question is asked regarding a particular phenomenon, followed by a thorough search for information about the topic. This information should include experimental data reported in scientific journals. Hypothesis – A possible explanation for an observed set of facts. Experimentation – A set of tests that verify or disprove the predictions of a hypothesis. Observation – A statement of results in quantitative form. Conclusion – Analysis of data Vocabulary Control – Serves as a reference; no change has been made Variable – A single factor being tested. Theory –Phenomena explanations that are supported by experimental evidence. Law – Statement that describes a phenomenon that is always true. Scientific measurement – SI (International system of units), is a universally understood standardized measuring system. Many SI units are units of the metric system.

12. Paula Jones pjones@homercentral.orgpjones@homercentral.org Dr. Susan Suarez sss7@cornell.edusss7@cornell.edu