WEBVTT 1 00:00:05.790 --> 00:00:10.759 This video aims to give you the basic knowledge about how to acquire material 2 00:00:11.059 --> 00:00:12.700 for further experimentation. 3 00:00:13.410 --> 00:00:19.331 But before we start, I think it's important to make clear why sea urchin eggs and early 4 00:00:19.543 --> 00:00:23.500 development represent interesting systems for this purpose. 5 00:00:24.450 --> 00:00:29.140 Unfertilised eggs are physiologically blocked in G1. 6 00:00:30.100 --> 00:00:35.981 Fertilisation triggers the entry into the cell cycle with DNA duplication and the 7 00:00:36.281 --> 00:00:42.121 first mitotic divisions, and mitotic division is rapid and synchronous. 8 00:00:42.322 --> 00:00:47.463 Sea urchin eggs are therefore powerful systems for studying the cell cycle in 9 00:00:47.763 --> 00:00:52.520 physiological conditions, as opposed to having to synchronise them with drugs, 10 00:00:52.820 --> 00:00:55.808 which is the case for cultured cells. 11 00:00:56.380 --> 00:01:01.222 The main objective of the first video is to show how to handle sea urchins and 12 00:01:01.522 --> 00:01:07.814 obtain gametes. We will show you how to set up a classic fertilisation experiment 13 00:01:08.014 --> 00:01:13.243 using one of our favourite models, the purple or edible sea urchin 14 00:01:13.443 --> 00:01:15.670 Sphaerechinus granularis. 15 00:01:16.730 --> 00:01:21.820 For this kind of experiment, we classically need some natural filtered sea water, 16 00:01:22.630 --> 00:01:24.500 a piece of gauze, 17 00:01:25.980 --> 00:01:27.950 a pair of scissors, 18 00:01:29.490 --> 00:01:31.200 needles and a syringe, 19 00:01:33.480 --> 00:01:40.290 we also need a few transfer pipettes, some 50ML plastic tubes, 20 00:01:41.260 --> 00:01:42.410 two beakers, 21 00:01:44.430 --> 00:01:46.300 a graduated cylinder, 22 00:01:47.460 --> 00:01:48.495 some Petri dishes, 23 00:01:50.690 --> 00:01:54.210 a few 1.5 ml Eppendorf tubes, 24 00:01:54.860 --> 00:02:00.239 a solution of Acetylcholine at the concentration of 0.1 molar, 25 00:02:00.539 --> 00:02:04.690 and a solution of Glycine at 10% concentration in natural filtered sea water. 26 00:02:05.719 --> 00:02:07.710 We will need a set of micropipettes, 27 00:02:08.380 --> 00:02:10.230 and the corresponding tips. 28 00:02:11.260 --> 00:02:14.340 We will also need a low-speed centrifuge. 29 00:02:15.200 --> 00:02:20.567 Now we need two sea urchins, one male and one female. 30 00:02:20.867 --> 00:02:25.537 Here we are in the historic room, called the research aquarium, where the animals are 31 00:02:25.837 --> 00:02:27.260 kept for various experiments. 32 00:02:28.270 --> 00:02:32.214 Sea urchins are kept in tanks containing natural sea water. 33 00:02:32.514 --> 00:02:37.556 For our experiment we will use the purple sea urchin Sphaerechinus granularis. 34 00:02:37.917 --> 00:02:42.370 The sex of the animals can be defined beforehand by producing the release of 35 00:02:42.670 --> 00:02:45.461 some gametes by a weak electric shock. 36 00:02:45.700 --> 00:02:48.870 Males and females are kept in culture in different tanks. 37 00:02:49.600 --> 00:02:54.400 After taking a male and female, sea urchins are brought to the laboratory where we can 38 00:02:54.700 --> 00:02:59.598 proceed to the experiment. It is pretty easy to distinguish the dorsal region 39 00:02:59.898 --> 00:03:01.500 from the ventral region. 40 00:03:01.806 --> 00:03:06.879 The dorsal part has the anus in the central region and around are five gonopores from 41 00:03:07.079 --> 00:03:08.899 which the gametes will be expelled. 42 00:03:09.380 --> 00:03:12.749 The ventral part presents the mouth where the ends of the masticatory apparatus 43 00:03:12.949 --> 00:03:17.557 called the lantern of Aristotle is visible. Around the mouth is the 44 00:03:17.857 --> 00:03:20.572 peristomal membrane which the needle can cross. 45 00:03:20.872 --> 00:03:27.317 We inject 1ml of 0.1 molar acetylcholine into the body cavity for the peristomal 46 00:03:27.617 --> 00:03:29.570 membrane to induce spawning. 47 00:03:30.450 --> 00:03:36.141 Then we invert the female on the beaker of natural filtered sea water and we have 48 00:03:36.441 --> 00:03:39.350 just to wait for a little to see the release of the gametes. 49 00:03:40.300 --> 00:03:44.658 If we observe orange cells, then we can be sure that we have a female. 50 00:03:44.868 --> 00:03:48.420 We proceed in the same manner to obtain the gametes from the male. 51 00:03:49.850 --> 00:03:54.220 We inject acetylcholine into the general cavity of the sea urchin, 52 00:03:58.750 --> 00:04:05.444 and after injection we shake it gently and we wait for little. If we observe a 53 00:04:05.744 --> 00:04:08.690 white or milky release, we can say that we have a male. 54 00:04:09.780 --> 00:04:12.445 We invert the male on an empty Petri dish 55 00:04:12.645 --> 00:04:16.310 and collect undiluted sperm that we called "dry sperm". 56 00:04:17.760 --> 00:04:21.310 This dry sperm is collected using the micropipette 57 00:04:21.580 --> 00:04:26.700 and transfered into a 1.5 ml Eppendorf tube. 58 00:04:33.420 --> 00:04:37.240 This dry sperm is then put on ice. 59 00:04:37.460 --> 00:04:41.100 It can be stored in the fridge before use for several days. 60 00:04:41.940 --> 00:04:46.960 At the end of this step we obtain many gametes that we can now use for 61 00:04:47.260 --> 00:04:48.620 different experiments. 62 00:04:50.460 --> 00:04:56.591 Before proceeding with fertilisation, it is necessary to remove the faeces or spines 63 00:04:56.891 --> 00:04:58.950 that have fallen with the eggs. 64 00:05:00.730 --> 00:05:05.420 For this purpose, we will proceed to a rapid filtration through gauze. 65 00:05:07.370 --> 00:05:12.300 A piece of gauze is cut and is placed on the top of a Falcon tube. 66 00:05:18.690 --> 00:05:22.360 The eggs are then gently filtered through the gauze. 67 00:05:30.500 --> 00:05:34.722 At the end of this filtration the gauze is removed. 68 00:05:40.680 --> 00:05:46.220 Then the tubes are filled up to 50ml with natural filtered sea water, 69 00:05:51.880 --> 00:05:57.330 and using a swinging rotor, the eggs are centrifuged at a low speed, 70 00:05:57.630 --> 00:05:59.651 2,000 runs per minute. 71 00:06:05.980 --> 00:06:11.708 After centrifugation, we can see the eggs pelleted at the bottom of the tube. 72 00:06:13.370 --> 00:06:18.020 And after several washes, the eggs are diluted to a final concentration 73 00:06:18.320 --> 00:06:20.140 of 5% of egg suspension. 74 00:06:29.120 --> 00:06:32.540 We can now proceed to the fertilisation of the eggs. 75 00:06:33.850 --> 00:06:38.540 Around 10 millilitres of the 5% egg suspension are transferred into a 76 00:06:38.840 --> 00:06:43.230 Petri dish, and it will be used for the fertilisation experiment. 77 00:06:44.270 --> 00:06:51.469 Dry sperm needs to be diluted before use. For this, 1ml of natural filtered sea water 78 00:06:51.769 --> 00:06:54.180 is transferred into an Eppendorf tube. 79 00:06:58.770 --> 00:07:05.350 50 microliters of the dry sperm initially kept at 4°C are transferred into 80 00:07:05.650 --> 00:07:07.420 a tube containing sea water. 81 00:07:10.600 --> 00:07:16.430 The diluted sperm suspension is then carefully homogenized using the micropipette. 82 00:07:20.330 --> 00:07:26.202 The diluted sperm is added to the egg suspension at the final concentration of 83 00:07:26.502 --> 00:07:34.453 2 microliters of diluted sperm with 1mL of 5% egg suspension. It is important not to 84 00:07:34.753 --> 00:07:37.423 add too many sperm to avoid polyspermy 85 00:07:37.670 --> 00:07:41.220 that will affect the correct dynamics of the embryonic cell cycle. 86 00:07:42.100 --> 00:07:44.270 Once sperm have been added to the eggs, 87 00:07:44.500 --> 00:07:48.700 we can start the chronometer, and add the experiment's start time. 88 00:07:51.510 --> 00:07:56.200 Fertilised eggs can be easily monitored directly under a light microscope. 89 00:07:57.710 --> 00:08:03.391 Fertilisation envelope elevation occurs within one minute after sperm-egg fusion 90 00:08:03.691 --> 00:08:06.510 and acts as a mechanical block to polyspermy. 91 00:08:08.300 --> 00:08:13.400 This even represents the first visible sign that the egg activation is mediated by the sperm. 92 00:08:14.760 --> 00:08:19.418 Following a population of several eggs, it is remarkable to observe that the first 93 00:08:19.650 --> 00:08:24.140 mitotic division occurs synchronously two hours following fertilisation. 94 00:08:25.340 --> 00:08:30.140 This natural and synchronous division is essential for cell cycle studies. 95 00:08:35.140 --> 00:08:41.530 At a higher focal length, this time lapse makes it possible to follow the first three divisions. 96 00:08:42.180 --> 00:08:46.840 Two hours after fertilisation, we observe the two blastomere stage. 97 00:08:47.590 --> 00:08:51.430 One hour later, we observe the four blastomere stage, 98 00:08:52.640 --> 00:08:59.550 and finally, the eight blastomere stage is visible three hours following fertilisation. 99 00:09:02.650 --> 00:09:07.329 You now know how to obtain and use the gametes for triggering fertilization of 100 00:09:07.547 --> 00:09:11.160 the eggs and early embryonic development of sea urchins. 101 00:09:12.770 --> 00:09:17.480 Before coming to the practical, please remember the three following points: 102 00:09:18.180 --> 00:09:24.562 First, the animal is easy to handle, and it is simple to obtain a large number 103 00:09:24.862 --> 00:09:26.340 of eggs from a single female. 104 00:09:27.310 --> 00:09:31.395 Second, eggs are physiologically blocked in G1, 105 00:09:31.595 --> 00:09:35.480 and fertilisation triggers entry into the cell cycle. 106 00:09:36.790 --> 00:09:41.781 Finally, mitotic divisions of an egg population are synchronous. 107 00:09:42.432 --> 00:09:47.145 This last point allows us to develop many different biochemical approaches to 108 00:09:47.345 --> 00:09:51.860 analyse protein synthesis in relation with cell cycle regulation.