<%BANNER%>

Developing a Noninvasive Method for Assessing Reproductive Status and Characterizing Gender-Specific Plasma Proteins in ...

xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20110112_AAAAAA INGEST_TIME 2011-01-12T22:19:02Z PACKAGE UFE0002851_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 4149 DFID F20110112_AAAATB ORIGIN DEPOSITOR PATH monck_e_Page_48thm.jpg GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
fc9cb2aa458db24257aad931f94c8df3
SHA-1
aff300fbbf64c2ce6c209114f0378556658826a4
58606 F20110112_AAAAEK monck_e_Page_64.jpg
6e126e494cc66580e9b505d216cf1841
5db3ece936ced1d8ac2d3d92e806f40517644e1b
7764 F20110112_AAAAOE monck_e_Page_01.QC.jpg
a986750763130dcb9a6980b444917cfb
f60b42e2d3d8222864aa13cdfddad63a90e4121e
50152 F20110112_AAAAJH monck_e_Page_30.pro
06e5162b8223676a1388b113195aba8e
55c4fee54c574130aca4ff5f91829e21abf1dc7b
39942 F20110112_AAAATC monck_e_Page_48f.jp2
0d63e76e798b03edf330550c93c72f26
6a86be0b8f5270fcb38fab78c58ae090048b5666
19109 F20110112_AAAAOF monck_e_Page_01f.jp2
0f36c824817a79b9283deb36091deeae
fb001903a906f62eba3db94875e7a1ad454cade0
47158 F20110112_AAAAJI monck_e_Page_31.pro
8621a53e392af8409e0ce05c3e2fb676
d7b8bdcab68ec234e076e29a3bff7b084ba69b1c
5735 F20110112_AAAATD monck_e_Page_49thm.jpg
26b42ecf138e28b0f4c4fd4ec0f19879
76d7724f0858a2162349361d9cdd71591e06af84
38880 F20110112_AAAAEL monck_e_Page_65.jpg
b4c96e4e2c5a5a88dd7a335345d3ae85
ddf6b8d483a54b5887c12976e26b373d63fac737
548 F20110112_AAAAOG monck_e_Page_02thm.jpg
a69053b7222835c7e633f20e97ef16ae
bc648861e4cc59d45ac0c5ed84c11a79879712b3
49028 F20110112_AAAAJJ monck_e_Page_32.pro
412059ca33214c6d13d38ec5d8eb39f8
e51323ade583d9a0fedf2eb8beed0f0d0550ef70
24278 F20110112_AAAATE monck_e_Page_49.QC.jpg
0cdbca72112f80d18d7ad821b0ebd1a5
118806b65e6deeb846bb52f0e37319551aa7bd9b
47782 F20110112_AAAAEM monck_e_Page_66.jpg
0fbaa7d676d6ad6a5a3221615c359510
b487481590ba7a905284163c4526970c78779060
1406 F20110112_AAAAOH monck_e_Page_02.QC.jpg
0c0b4abad57e7e09d28f203bd280c582
43aa48e8b83a8d1997a0b81f08c37b2aa5b04b3d
49925 F20110112_AAAAJK monck_e_Page_33.pro
dabf11f30fb094bde7704815068464dc
14c0520b6a33a1a491145179b4618221c56fe6bd
60397 F20110112_AAAATF monck_e_Page_49f.jp2
cac5bb86ca26d831fa22809dc74765e6
1e284cc530878deabf35cb04cb88d320ca2a8081
46344 F20110112_AAAAEN monck_e_Page_67.jpg
d3de007b16ca6a7ac9fa5250d6330dd2
c708b95243718fb43b49d6f1c47b099e032a394a
3442 F20110112_AAAAOI monck_e_Page_02f.jp2
227dc7ce47150abcd9987caae7301320
756b610eac3b0db5fad379082e61b85b1ce3bf43
49950 F20110112_AAAAJL monck_e_Page_34.pro
46cf2fbc319073c8c4c6df01936e5efe
4ac44770df7cf5891757e0c2f634783efcfc2bad
26973 F20110112_AAAATG monck_e_Page_50.QC.jpg
b2f8279735beac3ed4086ca9392c2347
719776d96b1dd44bec4d06b4f67ebbaeaf0bd43d
57410 F20110112_AAAAEO monck_e_Page_68.jpg
faeee42d8358c198e703df2c149db968
98b6f960a4b8da20cc7646d53dec798ea8d6d8e9
2226 F20110112_AAAAOJ monck_e_Page_03.QC.jpg
07d2949c7e1284bd82356de65a418923
ee990ce3bbcc0ad18b00bd1e29949952ef4f2170
50655 F20110112_AAAAJM monck_e_Page_35.pro
b962637fd3e22985233c30467287bfb5
c8d99fa5f43f7aa4c26057551f20f6c9fad62b03
66535 F20110112_AAAATH monck_e_Page_50f.jp2
9d76cc95bbe78f9948e08b12619c119e
9b34b46bc3cf9aa2dd40977b5daf9959701738ea
23924 F20110112_AAAAEP monck_e_Page_69.jpg
136d425b4705570447d6a710245442b0
8b59eb6593befb59556fa92f0fdc050cbe15e807
6822 F20110112_AAAAOK monck_e_Page_03f.jp2
584fa0f1a27362402f0d7f76e649bd57
ba686a6bf3de2ba643e73a972d11e38ef515e630
49108 F20110112_AAAAJN monck_e_Page_36.pro
313ccdc34c77e3b6eca7fa54cd9abe23
a3175880853092971b0dd18f5b221a15f51d8dee
21622 F20110112_AAAATI monck_e_Page_51.QC.jpg
efbc7be87f1bfa219c170d50a532b9b0
d3a3457ba1658687857842e9964a5fb0b52b9522
73336 F20110112_AAAAEQ monck_e_Page_70.jpg
60d91270deed71aef35daaa258c675e8
cbb2cc1037bf6f14252e65151d19d6c37a98b840
3584 F20110112_AAAAOL monck_e_Page_04thm.jpg
7a2835e67e72f82816caf652574afdcd
80f99c40ec8538404d4ff0669c7680ee9a4601cb
50566 F20110112_AAAAJO monck_e_Page_37.pro
0e37e427fdb16c0df54ecf3d35ceb21a
ccd89e50765602c80e5c407676ec01473135aa01
54297 F20110112_AAAATJ monck_e_Page_51f.jp2
9f75ce3bc36532fd2a9ba0ec2f042bb7
6770dd8d659e1580c14b4eb8f3e25f9f876f1b18
56475 F20110112_AAAAER monck_e_Page_71.jpg
0a4c0da6853712ec2def5a6429c4588b
7318c45d76ffa3d668a9b7f95a7fe97d18e02040
14073 F20110112_AAAAOM monck_e_Page_04.QC.jpg
6a36d4186d2131a1d7c91399e380867b
bdd631e0744dc488038395e915f4fafb6a662220
48860 F20110112_AAAAJP monck_e_Page_38.pro
c8dcad39ec8f5bfdf765f3974bae2c33
8c4d31114184fdc1e697ac2d7e517c8034a4affe
5978 F20110112_AAAATK monck_e_Page_52thm.jpg
5f6dfdd643c534cd982184a2fb12807f
ff23588c63c730c361c4f11fd6b47b3333e0f91b
77667 F20110112_AAAAES monck_e_Page_72.jpg
f49e9fd4089154208a4758ef8cba67ba
ce8c061671e346750c308b9fb85a687ac8391bf3
34849 F20110112_AAAAON monck_e_Page_04f.jp2
a0bf5860b75d9598a56927c02be91686
2d521152ec0b1ecc09fae1c12ec5d0c9ce27510b
24935 F20110112_AAAATL monck_e_Page_52.QC.jpg
51e9b2f2fcf198d7952320e2f98065a6
4204717581972666f81180811a0a6103461a4a4e
85856 F20110112_AAAAET monck_e_Page_73.jpg
b1d0ed093ec55a884be0488c34be71fe
4e8bc611ac62b946fc13b17a7e637a4c1cdfcd65
4548 F20110112_AAAAOO monck_e_Page_05thm.jpg
f84b10973dabb23d87a9fffeac0bc7fd
a5b57db1dbe56739ee2d1579b350bc56abc77294
45055 F20110112_AAAAJQ monck_e_Page_39.pro
fdc00f47da98956b6cb9807864def067
1c92c29e3902fb9a8a6876814cc186da4f2e9f70
60783 F20110112_AAAATM monck_e_Page_52f.jp2
d0b4ba23ac6dec824508164634f3f4aa
c620db0e484b40c76ab79530809b068e15233f55
28829 F20110112_AAAAEU monck_e_Page_74.jpg
f84d1dcfc3ce4122cd5c074e24aebb5c
24d8ac1d484bc98c2d83390a0f4c952d61b1dcdb
19788 F20110112_AAAAOP monck_e_Page_05.QC.jpg
e555577ec51d836376cc444b4e1feecd
2b704a2834fb7162636665747102fb2dd8a207ac
32174 F20110112_AAAAJR monck_e_Page_40.pro
854adaff7d18c50c316642011f98d050
c582e0693bea1d258b0c245180d37a8d2048eaee
6608 F20110112_AAAATN monck_e_Page_53thm.jpg
ae47df272296de6b7b2a10b266ae07c3
3b2d15ef40c398140054c8a958df5143feaa19c0
94324 F20110112_AAAAEV monck_e_Page_75.jpg
2ab1cd6b187b5f30454bf659ddce3670
b0c9bd1dc1854095cc175da4bc9cc91f24114b23
2144 F20110112_AAAAOQ monck_e_Page_06thm.jpg
b0003833511851258765d073c69dfddd
d61ceb60c482f787aaa6f2c5422148f5dbf37834
2258 F20110112_AAAAJS monck_e_Page_41.pro
6844fc2c2ba8d8de1c41db9be401339e
38397c49f63f96999d715c09d1a7a1785bb5a4f2
66669 F20110112_AAAATO monck_e_Page_53f.jp2
5b8fa5c4f1ad6d0ee6b1d64a556caede
ae2f3eb3d79577b40df1e5bca445887f4b878b59
111786 F20110112_AAAAEW monck_e_Page_76.jpg
6d340b0c9d5ae15561e08a592c935c77
dcac232914d9ca0c58fee90380d89d4472c4ff53
8307 F20110112_AAAAOR monck_e_Page_06.QC.jpg
0c7e0006dc3a54b8d3ebfcc40952b5ac
bd0fefc5fead7c0fa0c8e98a0466f884c5e20b39
6033 F20110112_AAAAJT monck_e_Page_42.pro
401cc0434d8a87742644ad5d536c3fd8
1ea48dc54a0a6b04ee771e8d8f2d588f2f34ee8f
5736 F20110112_AAAATP monck_e_Page_54thm.jpg
59f9a1ae81ca93049210cddb06f31455
3170584d95e169bc0fd2de99acf9ac0ee83955a6
109085 F20110112_AAAAEX monck_e_Page_77.jpg
d26a3437a0281b30bb5056d19b6723b5
432ecf662b9b8761788341cfb91b8fc1d735cb1e
442827 F20110112_AAAAOS monck_e_Page_06f.jp2
62fde66ecc5fb461c1639fc729254f32
fb4e29cef1f6d63aa5f528b8ff60aff0a0acd1c7
20881 F20110112_AAAAJU monck_e_Page_43.pro
18b1937eb5ea99d59de468396125fc5e
70d6737535e9353a4042756e29092804d8f6d0eb
22888 F20110112_AAAATQ monck_e_Page_54.QC.jpg
e321e50c2aed978c54f3577b8a72ce38
d3ccc1aa6e34bfdc633b05e569ebc1c04196566a
1672 F20110112_AAAAOT monck_e_Page_07thm.jpg
1a342522bc31d9a8699d0850eff3c5a2
6de6833b93816e5c544f02e062c6defe0e11f478
17295 F20110112_AAAAJV monck_e_Page_44.pro
ba1131fd136d480705c498c2ebae144b
b86bfb979e0c0287394edeefe09698a67131f1e8
77265 F20110112_AAAACA monck_e_Page_39.jpg
fc81285d918a1c243777de508795f33a
226c7cecb4c01721a4bb3e119c6322e7d80c9485
108889 F20110112_AAAAEY monck_e_Page_78.jpg
a8882dc56538d25eddf8bd3c64fe85a1
2cca10ad0910b7d6a7d1eea4f1010ef0a37e9ed4
57893 F20110112_AAAATR monck_e_Page_54f.jp2
ebe69767b6375ad9befb896b721e1235
d9f3ba0f364b8bb22417eef8fafc1e09fa1e6b87
236732 F20110112_AAAAOU monck_e_Page_07f.jp2
b3b464d961701113de49aa516932b60e
86a501d9761a128782656aa8a8ff905c1fcaded5
48717 F20110112_AAAAJW monck_e_Page_45.pro
a60143b2d487c3540487d3231c57b64e
e97f564eccc4a35b76088936b7bbfee07fc389d5
123017 F20110112_AAAACB UFE0002851_00001.xml FULL
f96a23f330aafd56dad89875ede0a9c7
38330d3d0398fd8bbe3f3bb4709b5325be498c3a
99977 F20110112_AAAAEZ monck_e_Page_79.jpg
3adbc16b8e732e813f60fc5b207b1438
d5f554437627eb79cb5a9ef6180dadd4dc210775
6324 F20110112_AAAATS monck_e_Page_55thm.jpg
77d5af16369747a96a91f45451a568d7
a731baeb2c7f7c21014e878c6705ee881124af07
50137 F20110112_AAAAJX monck_e_Page_46.pro
1a6e74a3635f12da3d9b5c47ac5ef657
fa0af7ecec0b084f58f32ba37d3b682b04630fbf
26139 F20110112_AAAATT monck_e_Page_55.QC.jpg
308f9cbdb7b270c7a86045be41ee7326
6cea4d3cc748c8318e2932f31a9a1da42f418896
3481 F20110112_AAAAOV monck_e_Page_08thm.jpg
267536d63875fa71b2e172405627a105
458506ff5eabeb440b4ca361b54578e3846a9a30
469262 F20110112_AAAAHA monck_e_Page_51.tif
5101090b0133256e3c5b8def8f8dc358
891c1de12f98f4bb023b62534e71a16652f11b15
52120 F20110112_AAAAJY monck_e_Page_47.pro
b8f82e0261ef04c09cad8dd8460f8c57
92a97f6a49a69f57701b39d60cd61ca255fc141f
65109 F20110112_AAAATU monck_e_Page_55f.jp2
665cb06f739744bc5b616ee20fa5f2e1
d1ffacec7df1da370c890e6fa1e6c6d3c3758195
14151 F20110112_AAAAOW monck_e_Page_08.QC.jpg
50cb437d30d98cc7e34d4b38fa369f00
ecf6058b511ffdfffb9768392d7105870c6754af
F20110112_AAAAHB monck_e_Page_52.tif
fe35501e59e8bf039edaa2d6754e81e4
207c0fe2ea8760faf5347ad1c380fbd01dc7a130
30146 F20110112_AAAAJZ monck_e_Page_48.pro
676df149fbe5af540a679151e2c62b83
0cbc6cb044a25c3306b9fb42e1fb1bb5566367b2
26709 F20110112_AAAACE monck_e_Page_01.jpg
1da4cee18c27d6950e6a354287e3b46d
0a804869f70990bfa1f3aa7f42f720012f9eba5f
6644 F20110112_AAAATV monck_e_Page_56thm.jpg
57f8eb7dd9aa2b4e2aafd2ef7a852cc7
231dfd368806f40be8705e6dc8292ca8aadfb13c
467580 F20110112_AAAAOX monck_e_Page_08f.jp2
9607f487a25ab37640e7e85effac5af0
eb0eb69f01be59e2946f8ae6deb147ebdac3ca33
F20110112_AAAAHC monck_e_Page_53.tif
26734bf2239443fefc0789e19fc2d46f
c9f16a986a6a99dc92612b31928d0a1a2d1a1efd
4278 F20110112_AAAACF monck_e_Page_02.jpg
de7845f1cd3f809172122d099c83f6b4
cbe552ad3f6017094fb5a1e7726a4c3edbfd65db
27466 F20110112_AAAATW monck_e_Page_56.QC.jpg
20a4fa031587ffc605f83ccfdaa9c803
89bf9cfa63165dd8e21caded0d703509ce659693
21529 F20110112_AAAAOY monck_e_Page_09.QC.jpg
31554f9fd2f6d13dcdf2f81f0bec7b28
5b648e26d769f08f174f0e6e9296a1eecab1328e
F20110112_AAAAHD monck_e_Page_54.tif
933c1d9c091ae38840080a1fefd185b5
89abf5956055df7c1346f6a2fa37cfda790a4408
9029 F20110112_AAAACG monck_e_Page_03.jpg
e3b57715735d39c2a318af023ecd6a84
b7bd54efdead0c5cac7ac5bf57423490eada9b8b
1970 F20110112_AAAAMA monck_e_Page_24.txt
dfef757ece73916879b3ee081d40ffb2
947671c38dd5c8a6abe68255baaa98f08359122c
65624 F20110112_AAAATX monck_e_Page_56f.jp2
04290caef257c714bb21be59da537556
3a91a19e040f2364729616cf38b1784e61328fd9
5984 F20110112_AAAAOZ monck_e_Page_10thm.jpg
a9ad9d5184add1a287e191c17c3e1610
310b5567b9c1230e5a5347dfba646379c0f1fe8c
F20110112_AAAAHE monck_e_Page_55.tif
9526de765500bb2b053beb9dbc733663
9fe047dedd883855497e83588a5eb1019710a807
45070 F20110112_AAAACH monck_e_Page_04.jpg
81a601a9d210d74ced9a7ba0c54f47e4
5318c72c4d0c9954ca21adb99204669b955208e5
2127 F20110112_AAAAMB monck_e_Page_25.txt
1dd447f4220e3e09fd26b83d43ee31c9
bb00e8a70557da9387e7ae3780293fedc105cdfc
26563 F20110112_AAAATY monck_e_Page_57.QC.jpg
b3eb198ab7aaf4fba7ac163368ae702c
e454855f50aff652bbf4147d24196d960baff43c
F20110112_AAAAHF monck_e_Page_56.tif
e5971ae0f8b76bc82e5da576e227328a
d97c51271823ee6314b699f8e89bf8ffe65d462a
86350 F20110112_AAAACI monck_e_Page_05.jpg
d0dcf220afb8347b3a6df811338d95c5
f6ec7fbd538c0d667f4d46b629078edc053e1ba4
1753 F20110112_AAAAMC monck_e_Page_26.txt
1ffbad6a0d1cf5be164bfeb567caa2f4
f051a6a5c2ffc39fdc44b4f2b7c6503c2498f029
64578 F20110112_AAAATZ monck_e_Page_57f.jp2
666285499000dc99967371c35f6634f1
950b539e18936f89c3240b30ea15d128c024ffe6
6063 F20110112_AAAARA monck_e_Page_28thm.jpg
5980b34ac394ae005c41a951a26e4b90
7b32bff0547a09d60ea8aae8b450af073c806a82
F20110112_AAAAHG monck_e_Page_57.tif
6f53cb8cf2373896c001100b91175364
b9d71cb64bf859e79fcce169739e64eeb742b02d
1915 F20110112_AAAAMD monck_e_Page_28.txt
cb619d00965fc6f4c90ae28ecb9bd5ba
defa4afea41ba9e17dc4cd9bb0ae458b625ba137
24983 F20110112_AAAARB monck_e_Page_28.QC.jpg
5f2bca94334d7e8801f53bce17a1b407
727b3278504f40984ec80ed0ef4e111eacf6e79b
F20110112_AAAAHH monck_e_Page_58.tif
3307286751e61f6afd910d76e93a049f
f1c7e5d267c25cfcd3619797863f80fdde8c20f6
36842 F20110112_AAAACJ monck_e_Page_06.jpg
7ab691bce9417d0403436b2d29b955a9
5d6be3ce0f5f9ef4f11445d72622851a007286f9
1984 F20110112_AAAAME monck_e_Page_29.txt
0436c2f09e2ba1e8419938e53db17b89
dbcdac31230a375d89625981e23f4da04a4924a6
62113 F20110112_AAAARC monck_e_Page_28f.jp2
cb2438320c57d51bd92269b42235a31c
48c87c890b04a9c827cb7c56f9dd0c08a0815e57
F20110112_AAAAHI monck_e_Page_59.tif
c9f25486a9f9acc68f45870943e0f7d0
c58e060e5150c67d9d5d056ef4c2cd7bd9ecb77a
20777 F20110112_AAAACK monck_e_Page_07.jpg
58c3687d9916ce76689893ea22100222
a1edc5125268046615016d5085661c21d03a51d8
1874 F20110112_AAAAMF monck_e_Page_31.txt
084268c056eb23aa5910e279e6f4f7f5
d42de4bd13bdbc4c1bf2fe023593fa4992a5627b
6825 F20110112_AAAAWA monck_e_Page_78thm.jpg
b980d2bb738ba0ba2d0a6e016d445c3e
344ad44ab076df1492e149e449fd026c94f3e1f5
6082 F20110112_AAAARD monck_e_Page_29thm.jpg
98a0476e7100ef9a76c1254d97feb70a
372fb7bd9678b63f15c3372be57ff6fc3aa46f27
F20110112_AAAAHJ monck_e_Page_60.tif
ff541b6dfa5776d0f298260877a799b2
d9cfc88932b11965745aea27ba8be48f0fdf2dad
49210 F20110112_AAAACL monck_e_Page_08.jpg
87a2a096f2db3cce996e70667df7ceb9
4342f3b0ce53848c7c450873bd085aa1b6aac477
1939 F20110112_AAAAMG monck_e_Page_32.txt
24fbf4c5526af854a182db7c411d17c3
295a00b46c9ee034ee22e491005be9a3abe6b49d
28738 F20110112_AAAAWB monck_e_Page_78.QC.jpg
726eee4c74c0240e07ac3fb322635b56
64508c7d35899fd4594b66d615e583ed95257b28
26575 F20110112_AAAARE monck_e_Page_29.QC.jpg
c9a4c8748d0dc7e1d5112d94580e11c1
3657da3d050a8af395c6cdb9a3053ed338cd15f2
F20110112_AAAAHK monck_e_Page_61.tif
58f44cc3649c95753ba5971e43bfdab5
a9b56c1798ca2fc12549e78a0b8aba395488b690
73228 F20110112_AAAACM monck_e_Page_09.jpg
cf42dc173709c66b91c90bce7421bbc2
3954b966d34bd915e67d6b782d0e72c484ba94b9
1976 F20110112_AAAAMH monck_e_Page_33.txt
00d00509a9c79c3f4e83c018fdd3cdc9
d299792f58a6be30ee01adf750693a8fac756170
83150 F20110112_AAAAWC monck_e_Page_78f.jp2
05b80c240f018390952362f3d01f8860
c1568d67fb2a3bf7113d35a472ea9d463a49acf9
64147 F20110112_AAAARF monck_e_Page_29f.jp2
6a142584b75b4dc52f2630cbe551ae43
d27d8b5eb48ac5d35f1debb8c13596e6295e9c55
F20110112_AAAAHL monck_e_Page_62.tif
6a38c83e5bd7b1d14aa8ae398b6b228e
56a4baf604833df5cebbea0abd5dd7bd45b49860
76739 F20110112_AAAACN monck_e_Page_10.jpg
ce7d48784f89b6721284bc784dfd7c8d
63d18a382b3c8760eacc7a516d057f2a8ba7fd6a
1971 F20110112_AAAAMI monck_e_Page_34.txt
88004c135754c94dc9ae4a1de2ab462d
6dc74d31f32ed5cd7a229352f23da676cd99e165
6504 F20110112_AAAAWD monck_e_Page_79thm.jpg
66240132cfc999919f7ec50911685307
1c8364dd271d44942a3bd329dc39df9b20b2b238
6333 F20110112_AAAARG monck_e_Page_30thm.jpg
c570f1d5185a30c423d66b60b1ba3315
eab7a64196a495af03d5085b854e83f012e3a137
F20110112_AAAAHM monck_e_Page_63.tif
96450046aa7a1d9b0d779ba36aa2cfb7
139301c8daedada7d5f5aea9977effe35712f95f
72994 F20110112_AAAACO monck_e_Page_11.jpg
dff284795fbaa6233097c20f0e592bfb
3876dcdd8b222d8a0b8ae68606a86509189dfd52
1989 F20110112_AAAAMJ monck_e_Page_35.txt
c63a88ea5d7ed79965061fe9c4c3bd97
bda7e183d4560cc7f9a225a62ac5674ad3eaa465
77595 F20110112_AAAAWE monck_e_Page_79f.jp2
0d810c7ffc405a010b9e20925ac0937e
56117078eaf81ec8dda8d4d1381582aa33b641b1
26265 F20110112_AAAARH monck_e_Page_30.QC.jpg
80f2bc011712f043cbae6f2e10534adf
4b9ba0e5e46e6ce2b027595bd2b2b768bd9be655
F20110112_AAAAHN monck_e_Page_64.tif
a98ae869c27e818f3540600b13beb865
9297e18b0b3362f20e44142e1ad039c4e654526d
84904 F20110112_AAAACP monck_e_Page_12.jpg
e5f828addea3839a99d793ef37f06b93
2735431c70e86a6ebde5e5e71155a1a963f25fdb
1987 F20110112_AAAAMK monck_e_Page_36.txt
11fb0d34aa1570ad691594188960565d
e1816523bedafc6a7b42858fa08a28e173e5dc58
6445 F20110112_AAAAWF monck_e_Page_80thm.jpg
1cecf8db2e6d7fc6e025dfb186b4affa
7a22294208739038e09b1d70b2d14b068892377b
64488 F20110112_AAAARI monck_e_Page_30f.jp2
8cb30df3525bf3a1916f99480cfdc3f2
580c791e7ce1cfb2488acb5b135d19b7ab0847de
88303 F20110112_AAAACQ monck_e_Page_13.jpg
134f6900768099e18230b25c03677c69
4a539ae1065fd36654d24200d39e45cbba57da6d
2000 F20110112_AAAAML monck_e_Page_37.txt
8b377acaf14102fdf297004a627c9363
d552b97264c2c113eef0351f1e16b78c996dff66
27284 F20110112_AAAAWG monck_e_Page_80.QC.jpg
b46eeba3746f77efe5ab1a912334b9fb
c0869aa844dde8ce4e3b7ea5f6167223da1e243f
5899 F20110112_AAAARJ monck_e_Page_31thm.jpg
c894d7a86fd8d5c0b2f9c3afa316d8a9
4c0f4a0c83483bd7e81082c9afad35593cf0ab2e
468170 F20110112_AAAAHO monck_e_Page_65.tif
3fa95821a0a22c5602ebe36d7b64994c
638ba3874f454794dbe22e03e9e0cd374835d928
79608 F20110112_AAAACR monck_e_Page_15.jpg
5d82e41fd4ec73d237367e1d930d6629
0292368aa9b705bbda505f02bae71f7402513a72
1928 F20110112_AAAAMM monck_e_Page_38.txt
e0e742e2b72f2798577fb38359bb77f7
ee47b344fe72fec41474efbca2a822e35ad8baf1
78127 F20110112_AAAAWH monck_e_Page_80f.jp2
e6fe1d2fa35ff2282e8208e43d0221ad
9207c532e9e221c6bd8e2af125fec51e218d3f11
25245 F20110112_AAAARK monck_e_Page_31.QC.jpg
99b236ddf3b26c6c19cb92b73c95db80
433461f6b18924c28256d6b467dd9e545d75f996
11237804 F20110112_AAAAHP monck_e_Page_66.tif
8714d889b5ab3cee70ab6cbc52c822f2
1de88c5f067836b1e18c3a2d507e9155914a50a8
81642 F20110112_AAAACS monck_e_Page_16.jpg
4627e13a2f78bef1e1ebfdb4305966e8
54c40a880ba5af495551a96bef2157b4eee4f36f
1467 F20110112_AAAAMN monck_e_Page_40.txt
6c71984c07c6f5134e741638e3876f67
9dc7da94eed8ae1c5bc6778626632136c9de585a
6689 F20110112_AAAAWI monck_e_Page_81thm.jpg
746bf3208d6b764b54781d86cf583c45
abba8c82150d8cfd463fe6a6024ce2a7dca68933
61631 F20110112_AAAARL monck_e_Page_31f.jp2
b50a6a429c5f24a048da80a431f32a14
1123e4d85ccebef1a9ba31b633a70be6126dbac6
6318054 F20110112_AAAAHQ monck_e_Page_67.tif
f7fea41d237d7a314625bf1575acdaa8
a99aef15098ed9e9343781eac0f11ccbb3e093db
67956 F20110112_AAAACT monck_e_Page_17.jpg
eee7497cb770ff4df15c22f09f788035
cc73815b462d9e1d9ab8a21484ebfb828f970c44
176 F20110112_AAAAMO monck_e_Page_41.txt
5aed5c0870c04ebaa32264bb978671a8
b6bd36ea5d168de2221d49020392a09bbab68e01
30409 F20110112_AAAAWJ monck_e_Page_81.QC.jpg
0c81a5b97377fac4c17a56ae75a96e8b
7d3175679e3f7062330c72f9828cac06113fa3d7
6437 F20110112_AAAARM monck_e_Page_32thm.jpg
a77516622f5105deebbf670fdb4a028b
e75e7c5a313131241351f445eaf18d21835b5da7
F20110112_AAAAHR monck_e_Page_68.tif
5b20223da31e8a36a7c28be794e66a91
965002287b4f66a7f2b2b1b2825d674056bf149f
84715 F20110112_AAAACU monck_e_Page_18.jpg
7cd2552f70d5c2a27d3cb42ab0f01781
205ffb3446b921b5e0333f3badbccc557419f9e0
322 F20110112_AAAAMP monck_e_Page_42.txt
90995aa274c424ec267619b7eaae6be3
dc451aa61d4a7fc1684e95e3efba1dfbe5403584
88572 F20110112_AAAAWK monck_e_Page_81f.jp2
003e869876eed16d01c5fe66f16e86a0
4b227bcab31dafc43f2344803c143ea6fe1a0275
26638 F20110112_AAAARN monck_e_Page_32.QC.jpg
c4b0da2418c3124afe5b7bb68882c7bb
59759aec3a4ac6c5eb400feb1b01df5dfbd22909
F20110112_AAAAHS monck_e_Page_69.tif
d6f8ea94f296945a24caa5485c600b43
dddfa3ca4f84f8ab2280d275b526d223fa7e452c
92275 F20110112_AAAACV monck_e_Page_19.jpg
117c026376fb771e09bf29a1bd182e2e
ab1c11960fb0430c1c36380211b3da39f15372ce
825 F20110112_AAAAMQ monck_e_Page_43.txt
7942d16e525cf3fe7949ec79d6a26a5f
949baeefb533459c85b319c6209ba008c587276d
6821 F20110112_AAAAWL monck_e_Page_82thm.jpg
46340c02513409b2b6446e24ba10a74c
574a19842e554f603e4892a538bc17b9db3a84c0
65552 F20110112_AAAARO monck_e_Page_32f.jp2
0021e203fa50eeed464f0792665d8ff0
ab0710a0cbbb7ee1e5cf1b3797103d9dbb51fd3b
12388734 F20110112_AAAAHT monck_e_Page_70.tif
dacccef04da6ca64058ad32603bad5a8
5401a18ab4dccc0eaa4336b77771edc7629bce4f
82799 F20110112_AAAACW monck_e_Page_20.jpg
cac3b388d34fee60299adc7575106856
78d4b616f97d31466c6a08424a51264aef666c9a
766 F20110112_AAAAMR monck_e_Page_44.txt
97e5237c86ecc690e15a2d4074f655e3
db11079249f0ddc6540270e1eba02111ffacdc9e
29896 F20110112_AAAAWM monck_e_Page_82.QC.jpg
5e963d4ba2560d63ce233759cc14b6ca
7bc4f1f9638139fecc471a2a014066d6cdf91e98
6457 F20110112_AAAARP monck_e_Page_33thm.jpg
355e63356513ee8cd66a0ffff989d52d
f766795a53e13350d70a602fe78fa20fe8c9de82
83628 F20110112_AAAACX monck_e_Page_21.jpg
0c90e03ad14538118e0cfdcbc99d140b
2919b098c18f13bc3ab575fe1c6f2b111dbe1704
1973 F20110112_AAAAMS monck_e_Page_45.txt
869f6f861f0f0dbc81ee5fdcb6ec597f
5778d8b846fc7a92a9cbcf2c62b6ba806aace344
F20110112_AAAAHU monck_e_Page_71.tif
b4f520a6681eb51b22a2cd5ad87ae18c
672cef747e1c5d9da341a8287a5d715e42e475a1
83769 F20110112_AAAAWN monck_e_Page_82f.jp2
3f5d48b1b9d3e6813485933c5ad6feae
5a2d8640904c300deac74f812f237a5cb08d0203
27197 F20110112_AAAARQ monck_e_Page_33.QC.jpg
08f35b7f535b9f3861dceba6ac94ed5a
5190639c25fd3c315c50a5def55b9ef4560e9b98
6426 F20110112_AAAAAA monck_e_Page_37thm.jpg
0b9f69837af25ec7714224a062e6dd7f
954ad88e7e6d8a5d1e76613e79cb6449ab1c69f3
84680 F20110112_AAAACY monck_e_Page_22.jpg
4171d2158129d65eca8697550449d151
d35ecd3ddf67af00e8bd49c65fadf03b0f7ee073
F20110112_AAAAHV monck_e_Page_72.tif
a9a642f3a5b389b41f77b159c04daa2b
a049c3b7acc8584efb664853675a159d1737a6d2
2481 F20110112_AAAAWO monck_e_Page_83thm.jpg
1a7c3bccebc200b8bcfdc4b16df052f3
b4c0dfeba687b7c53ab37f9125d2b629716d46d4
65730 F20110112_AAAARR monck_e_Page_33f.jp2
3826459e1bb14f9344c3938eb2b8b00f
4e83010c992901f5833a20bded4d5208d5b52974
3364 F20110112_AAAAAB monck_e_Page_41thm.jpg
e90013edf40cd1634e57c40d350b181a
17617d124396f18ad0f7927c92ff76a8949d3f48
81908 F20110112_AAAACZ monck_e_Page_23.jpg
a3743d934096ba16790294b9c27ab00a
1a5793f7239cbdba976c29afcf1c25b54a984c7a
2047 F20110112_AAAAMT monck_e_Page_47.txt
98c8240afac2204a69992e00a36452f5
437d220bafd194d38a6cc8f056be3f68203299b7
F20110112_AAAAHW monck_e_Page_73.tif
d0e69830f3a4512ee4d39681207fbc1b
a9c14f1a8c20c10d85263d8e71bff06e849f0112
10649 F20110112_AAAAWP monck_e_Page_83.QC.jpg
fc3de60890e3457ff501883e5434955b
0fcc10b29521f344d12872a68a3da4ddcdafaa5d
6656 F20110112_AAAARS monck_e_Page_34thm.jpg
42bb59f842399dbdc0fb8f83edf833e0
b3a604c697b0324a6254e556d1a938fcba2fcd0a
82930 F20110112_AAAAAC monck_e_Page_24.jpg
2a5c086e1a0d6ccad75845500bf0cec6
93f17eb176c7b1e7d0bd60059491b6921a50a522
1204 F20110112_AAAAMU monck_e_Page_48.txt
6ea1193b02d7398f434f9c43ebfee2a1
fc0c80dcd04f55dacc861a9d3dbd37ffde1059ae
F20110112_AAAAHX monck_e_Page_74.tif
a95d250a217e75f67d25f6c58f3a4119
b317d9f0b5d78ef7ad858b2c2d56c656a315b6e5
29070 F20110112_AAAAWQ monck_e_Page_83f.jp2
86cf6f50f3aa2eefeafa61cc039cf131
921390e382b5878be52d8fe645d5dafd06e5d9b7
26423 F20110112_AAAART monck_e_Page_34.QC.jpg
15db9b7b23202a9884a6be99e50a3ad5
97f4bca2650ff4bdcfe9e96039b970cd69af6f0e
F20110112_AAAAAD monck_e_Page_22.tif
8a56a8d176e08b9f7351bd802afb8471
1f0b0d18e8f13939e448c629326e42b86aca8145
1837 F20110112_AAAAMV monck_e_Page_49.txt
10cd5cbac12dffd131f1262f6c3b0c33
79649fa08a3bc2b69519051b36ac1460a37d24e1
97682 F20110112_AAAAFA monck_e_Page_80.jpg
ef04aa661b9c0211de99a6a0a40c4de3
34bcfca70b01825b61ce01f02f8a4b805536b03d
F20110112_AAAAHY monck_e_Page_75.tif
ea95cba387ba1aa4de230807a8987377
f9edd0f43b9663ee1c75e31c1141adab6fde29aa
4542 F20110112_AAAAWR monck_e_Page_84thm.jpg
fda98b816cf10c26ce7d6147c885f030
f4fec21acc2742cc8209f38f85bc82e2a4700ee6
64877 F20110112_AAAARU monck_e_Page_34f.jp2
deb59883e733130f7a0f2bbcf4986fcb
835952088720ec3f58ecc0eee0eeb00d5a4f1c46
20350 F20110112_AAAAAE monck_e_Page_43.QC.jpg
3d07bbd781f5629a66affe3c16f2d777
6e2fcc122b8e162a88a04c1284b8e89b2165be16
1654 F20110112_AAAAMW monck_e_Page_51.txt
af534a24c1788ba7799e0023b7b3ecb7
ed22421069d3deb6ccc2ecfcc43e5546781b0cb5
114532 F20110112_AAAAFB monck_e_Page_81.jpg
27c9aa41b36a21cd9f4e0c4cee9f46a3
8a3a5c7865f0759638edb6f16612e066781bd82f
F20110112_AAAAHZ monck_e_Page_77.tif
eff95eed92377b813a5ef8da2d78645e
9ce6982af69013a3bdbc1d5a679cd9c199e8222c
17763 F20110112_AAAAWS monck_e_Page_84.QC.jpg
7eb2624033b654689b246bdecfb14daa
6cf41e7b6eb12b64720743194b8a426dd3d6a20e
26815 F20110112_AAAARV monck_e_Page_35.QC.jpg
a1cd0e8c99cbf924cb53806bd8babd18
aed8b2d3972f7c9114e686e99867505161656bbd
68808 F20110112_AAAAAF monck_e_Page_81.pro
71e118b5e672d3c664e0ac46caf5f5c7
a528e9bed5d1050b117c7fdc4cd89a3f8de5825a
1816 F20110112_AAAAMX monck_e_Page_52.txt
50b9a087bc489eacfab542111893078c
e05cf94dbccc5447b7d4541a60c74654c7e163c6
111855 F20110112_AAAAFC monck_e_Page_82.jpg
a06bb81ca7ec4c40ebd7debc2407cce6
f6062bb21249e0708f93a00dda6f877960224bdc
44440 F20110112_AAAAWT monck_e_Page_84f.jp2
9c064a2547ba2074fc240be256245ba1
0ff99020823ab6184b3c0e60e828720bcdf0ec65
66100 F20110112_AAAARW monck_e_Page_35f.jp2
f13a239b53857ab1241d3c7a5f1b18dd
f6b64ce6adb8e848c20bdbc3dc4a2890dcf22e19
56365 F20110112_AAAAAG monck_e_Page_09f.jp2
ee5e035e6bc2cdb753f2cc999291522f
d77a19cb77ff5b5884744e4cd6c5383a7e2a3859
45087 F20110112_AAAAKA monck_e_Page_49.pro
352f93cf72b21be74562e30607b227ea
ed7f8c924b937ca1a737a58fb2eea4cbbd0104a9
2015 F20110112_AAAAMY monck_e_Page_53.txt
66748f0526e1410ea83c2eb64ad07b1a
465a789b5a2076bbb0c96024267fcc6026431044
40074 F20110112_AAAAFD monck_e_Page_83.jpg
23291239815f67336bdc3f8ef22f08c4
75a264aeb5ef7a6483fc9099355831bbcb1c6067
80572 F20110112_AAAAWU UFE0002851_00001.mets
0dfad8f2837400b0de7b587e1b9fc979
f7faac7c08ed6452e5d20be5a72eb5a89e6a6f44
25948 F20110112_AAAARX monck_e_Page_36.QC.jpg
16c4ec98883fdc68d7c863db7a77c1ef
36e3cd900f40bc71bad87dc98aa990c30909289f
51623 F20110112_AAAAKB monck_e_Page_50.pro
f6c1898795f84b1375288bd932593542
04678c07d3774363e28d44883ae62f31c54dc2eb
1702 F20110112_AAAAMZ monck_e_Page_54.txt
b888bcbcb4a2c53daa06399318de72a6
a57f28d003b35686ed7ca015b03632cf582e50bc
56210 F20110112_AAAAFE monck_e_Page_84.jpg
e681237119777b286e3f098ff4c1d53d
9254e088a0e303ee4eb440fbbd7f0a139b50983d
40546 F20110112_AAAAKC monck_e_Page_51.pro
d287c17784dbd228a50914537247ffec
2b09f4404d2cb9cfb2d307346af7cb1938ab5073
F20110112_AAAAFF monck_e_Page_01.tif
a0eb304fc3649d9f2e46c73dd10112ef
ba37d89d63a85774e6a888da9d08e381668d3c2d
6588 F20110112_AAAAAH monck_e_Page_14thm.jpg
1a112ebe42b6d6ccec90752dfb8bea05
c02532b7f98a8e58b68843ba640d0240c518bf1c
63443 F20110112_AAAARY monck_e_Page_36f.jp2
7d4229357d4a80c240cafe0a432f6150
8c7acd1981e07f00724416b2cc8fa035834508db
45403 F20110112_AAAAKD monck_e_Page_52.pro
1442f8f98917cd307d11f54f1fb1dbd1
a17780fc79de00c42810967bfcde8e77aa51e51b
F20110112_AAAAFG monck_e_Page_02.tif
9f6c8a28f9ab1f668e5684d9a02015b9
80d088b0591147ddb11f73cb421a62e4854b6258
23866 F20110112_AAAAPA monck_e_Page_10.QC.jpg
13828f3500d9a4237bc393e88011071b
7e7badf0c7ef15113e7ccf90c14cab8ad9df3656
5244 F20110112_AAAAAI monck_e_Page_09thm.jpg
7f1b26db56f58fcfb0743af158873051
21a4359c622f3c54d35b829434cc40fe8beb3141
26582 F20110112_AAAARZ monck_e_Page_37.QC.jpg
6a46dec4347cbf737008f5356cba894d
39f8a32afa65e6386f4c1923f1c5e84b2afbbb61
51320 F20110112_AAAAKE monck_e_Page_53.pro
1a92dd72cdb2a064fbc01d7ef078e385
504439b5825be6225163b715f38b7f506157da20
F20110112_AAAAFH monck_e_Page_03.tif
2a0acf5069cad30d8b61e9cc2f1b03cf
d4baed6bcd0e9347fa4bb5399914d2443b859cc3
59952 F20110112_AAAAPB monck_e_Page_10f.jp2
b3ca475e230d28c30fc88645e1e3bcff
fed0cbdf973faa8675a9298cdebf6fcbd181d600
19914 F20110112_AAAAAJ monck_e_Page_66.pro
33a35d035b2faa1a5bfb5a65a40768af
f14b3c7f3770895d708be76c915f816f93bd2801
42720 F20110112_AAAAKF monck_e_Page_54.pro
1f7570e4414eae49135e20e262aadaf8
c0e4d1edc02b332648e141b1d11b732bc9746e04
F20110112_AAAAFI monck_e_Page_04.tif
5f4c2cdba8b7ad22e786fc194b2b5d88
9b5bd984a9f1c513d6b42ee1a15b5ad62f8671b1
5609 F20110112_AAAAPC monck_e_Page_11thm.jpg
109baf2b28e4d3fd28bd73c84326ff7d
13f68fbe1d0e089abf5865ebbeb5c3d88c4e1c20
7958 F20110112_AAAAAK monck_e_Page_69.pro
1b2a3a91dbd7192dfe1f3d47f3830893
cde53f0b3d2435b5b10553808d3a6daa515fd091
6393 F20110112_AAAAUA monck_e_Page_58thm.jpg
fc791f44a3590a10549c5c54929a1639
5ed621dab6d254e82ffbdb6c85053be421e7ba5d
50220 F20110112_AAAAKG monck_e_Page_55.pro
cef19915ddd7b9db86a2aa086bf32cef
3e89f39c58be3417583840a9e1521bff35b11781
F20110112_AAAAFJ monck_e_Page_05.tif
95de9f1b73453ba028f44fd6d0f37200
cb4ad51a4a56e81e226e36f9fc7f7a2952f9434b
22764 F20110112_AAAAPD monck_e_Page_11.QC.jpg
9f42dbd80fdc6ef650af54ba008e2d9c
e09d75314ba0251d339823574af6908b56f087fe
2470 F20110112_AAAAAL monck_e_Page_77.txt
1ce1b7cd528cac799377b29e48e17a8b
c66b235a877cc98dc6943621fdaa513629d50c8d
26408 F20110112_AAAAUB monck_e_Page_58.QC.jpg
13fe1863eb6fcbdc5bc839f1fdc5e21a
ba6185c719d49ff2b5e3828f83373f837be4a9f1
50406 F20110112_AAAAKH monck_e_Page_56.pro
ae499e251b31ea5db9432e57abcbe0f8
713c70a4491049ea315e7144684adeeb0d3c1d1c
F20110112_AAAAFK monck_e_Page_06.tif
20ad5124294ba3abbf3b6d80362280e6
ddae162639c6f4b3a4628af364728e522651c29a
57374 F20110112_AAAAPE monck_e_Page_11f.jp2
3b58a30090b4922182073d96ad3caf08
0f1d14412e10d414f036c9e35c1a0437d953b3ac
50385 F20110112_AAAAAM monck_e_Page_20.pro
46eac7f92a84e3f407fc86ed4727b914
37a488c4249c0c8377ee65fc17a0dcad7f062578
66608 F20110112_AAAAUC monck_e_Page_58f.jp2
5ba95ed0ab9c9dde48d43800eaae33a7
58ed593dda024daabec7d58322004f72b71e90fd
50214 F20110112_AAAAKI monck_e_Page_58.pro
0b36db05968d1e4a3da6af6e512015cd
339bf6f9fc4b6f246bfd04dbee1ccc2e60f8d5fa
F20110112_AAAAFL monck_e_Page_07.tif
345aaa1e49f8c7a9d4f70ee2af1a0916
842a8b2ee7a4f38cdf1eaa37b4d8c38ba1ac6ed8
6498 F20110112_AAAAPF monck_e_Page_12thm.jpg
7dbd1e5eb754b4e7efc229403482585a
e825ba1b77732428ebd08d82af443842de9c579e
26271 F20110112_AAAAAN monck_e_Page_63.QC.jpg
833ddd1afbd6d6172e3215c2a0d2477a
58713f93d50c5909fd33549b5cd85a4c834567c1
6549 F20110112_AAAAUD monck_e_Page_59thm.jpg
fdb9ecb05bc72c4d1afed7be925eb91c
675937b4421bcbccba603166315e19e295556726
49644 F20110112_AAAAKJ monck_e_Page_59.pro
9414f8de44ec02545825ddc8e1804dd7
d9baf9d319fb28233ad2b70d557368bdbce647c8
27383 F20110112_AAAAPG monck_e_Page_12.QC.jpg
a4d0725cd21e153d0a3dc3b1c8a828d4
fb7ab57a162fcb444926bcbb7202fb4d83f69e39
50416 F20110112_AAAAAO monck_e_Page_62.pro
5610000673ebd2add95f89bf7b43be5f
19e321d878e0cee162427cd55fd6a4c71212cf5c
26579 F20110112_AAAAUE monck_e_Page_59.QC.jpg
c021640a21d57db11e120d7104e60ec2
3f970772c326b132cfa3dcd8e41a8041c2a1d240
45340 F20110112_AAAAKK monck_e_Page_60.pro
2a1265d26ece5de237a739878a9665ab
df0cbf078586df77422de84fb31fcdf6035508e7
F20110112_AAAAFM monck_e_Page_08.tif
98ed92cf6fa1b0c82b330777f50c6cca
b0c4f44ea1c5ea04c34441221c51b6b33179c486
67496 F20110112_AAAAPH monck_e_Page_12f.jp2
9b827d1c63cf2bd3fd9648375383db8d
2fee0d027446b0bc093bd271c501908cb9a63505
2032 F20110112_AAAAAP monck_e_Page_50.txt
fa408c4b2b6cf8a551eadb8189961466
06323b8125f0c48199213977641c70c3b28e9e6a
64836 F20110112_AAAAUF monck_e_Page_59f.jp2
8a918203a926df3ea06286d9ea68eb7f
144ce24e93188786614ace31b819e021c4cd5ff0
48840 F20110112_AAAAKL monck_e_Page_61.pro
8630f1ada8d31dd13a540fd397d632b6
0408cd4f7745cafe814b0543a61bee81f5f60f5b
F20110112_AAAAFN monck_e_Page_09.tif
59d02562e5766134fa577651519210f5
0ada57bec0c7ca7445a033f3d51349b3808129de
6679 F20110112_AAAAPI monck_e_Page_13thm.jpg
682ddc10d507cd3422c8e55234dd833e
4ed54f453dc9942eb77689da6dce3b97d438f1f0
F20110112_AAAAAQ monck_e_Page_13.tif
6ae931edfcaf8d5f9eaa5c459d8907b9
529024a22da6a3583bc7fd7663019e482100f0bf
5943 F20110112_AAAAUG monck_e_Page_60thm.jpg
2d005c6a98b157741a4df4a94af3cec9
8560bb7834dfa75d9a4ec131bfe3980b2f906a04
50748 F20110112_AAAAKM monck_e_Page_63.pro
ff9ea895ec88bf157e36a0b404b2a049
992f288658869d89d2a79750745b79edf6b11ac8
F20110112_AAAAFO monck_e_Page_10.tif
db2d5e2933eccb120b48cf242488885e
74b2259e6618ea5279df2d078f111cd2de30d8e5
28242 F20110112_AAAAPJ monck_e_Page_13.QC.jpg
0ec8c970b3bbdd326e97bca22bbdeced
15170e0b447749c0ad9a5a9b84fed9c28f9713ad
1366 F20110112_AAAAAR monck_e_Page_64.txt
46eb8ee7be3519380c17e8d11c73b94a
ff954dd98648ea60224a9d4cc4f39bbca3c8c64d
24142 F20110112_AAAAUH monck_e_Page_60.QC.jpg
ce74012cbe2983e5f1f7b18f6ffba391
0db16237b8da3aa6c5901ebb8eb48c992399ad2e
39468 F20110112_AAAAKN monck_e_Page_65.pro
edeffd577d1a467b73e0cd203e14077f
40bc8f8a4476e3f4b9b8688a3d1e5a0bfc4bad4e
F20110112_AAAAFP monck_e_Page_11.tif
4a3a1da9c34c649fce86cc4d5b057700
75eb09e3cc15fda52dbf5ab35fd6a5653066fd31
68549 F20110112_AAAAPK monck_e_Page_13f.jp2
8e5f8f66520043dbf167a1ee6511309d
9be458406c7ada3775aab57dcf9fee25fa825224
27719 F20110112_AAAAAS monck_e_Page_53.QC.jpg
5aef57bb2a14d31e9531a8051aaaa4c4
6de5166a7b13d51f5f49cdc439da3ccd5b5379c9
59691 F20110112_AAAAUI monck_e_Page_60f.jp2
8539853a1bbba4a270e40099f056f28e
8424ce3207229c2e2eedd9e6d7a80a0a11159142
18728 F20110112_AAAAKO monck_e_Page_67.pro
3e2bf02664b9348a2b1e347c6b4f972c
3fff833cbc6cc1203b2dc37e4ca94778d22c40b2
F20110112_AAAAFQ monck_e_Page_12.tif
9f36f6c42d18e88c806c2131402f7ea9
c8118dc57b037f3f1b607485238df8db1f2ddd64
26418 F20110112_AAAAPL monck_e_Page_14.QC.jpg
fce44203caa2b108b8b6c715243faf1e
0712cad44450e9e200161e34b55773ac32930f83
83899 F20110112_AAAAAT monck_e_Page_14.jpg
e04ca54ad027e23a27d9d13ee6c326c5
f4bfd1517b6fef0e24ed7c30e5936cc7a7418d31
6256 F20110112_AAAAUJ monck_e_Page_61thm.jpg
f8889e6f53451d4422965c3cd01517fc
9ad3c3519c430f7365f8cf96c6a60ecd7ea98a98
64425 F20110112_AAAAPM monck_e_Page_14f.jp2
e5c901fac8891cb2b4d468d65b02b8be
fa4396381ad3fa6912a9f5844e425e21d1527bb5
21355 F20110112_AAAAKP monck_e_Page_68.pro
76d3e1d96d1e134e044753b8a7bdb0f7
84a1a7b1c3615bfb1deed09a2059c48330941da0
F20110112_AAAAFR monck_e_Page_14.tif
fa1a1108e8dc5b0765af249c7744d4f3
89a1d514caee7ffddbefea76c9c429976f839cb8
1804 F20110112_AAAAAU monck_e_Page_27.txt
e26e6ff95ed3dbc1b2ddda1244bc9c6d
98e5a2d32c74e3ec57445c7da98acb0c8012eb8f
26168 F20110112_AAAAUK monck_e_Page_61.QC.jpg
8a0f860408b20dec0cab7e1cdfbab049
a53899455e5a8117cfd2a797c71adce9c092264d
6213 F20110112_AAAAPN monck_e_Page_15thm.jpg
1bf3832c4e4dc3ddf4aac1ee805eba8c
c5f18522747d65cefd100158869b3cba8cbfe717
23802 F20110112_AAAAKQ monck_e_Page_70.pro
2357338dca08c8a2a2e659194a288ac2
95350655987694d20b87e91d30a5a9bc249bdb0e
F20110112_AAAAFS monck_e_Page_15.tif
a61a6e6baa80379039a011ac4c304561
8f1715bbf469df5954a5fb4d895fb437d46d5988
34414 F20110112_AAAAAV monck_e_Page_64.pro
ec0bed988cb3cecffb68e55cdbf3dbfa
0a3f9ba30cebc3e2c5a2d6e6069d3d54b8e35d09
64154 F20110112_AAAAUL monck_e_Page_61f.jp2
792ca9dbe58fbc8c0016f7dc0605a68a
2b8669eafb8f580f7daa5e4053c312f114207360
26000 F20110112_AAAAPO monck_e_Page_15.QC.jpg
0ef050ddecb017236d17d039578362bc
74d6f7264e7c258fda91a9fa39abef765ab80e13
F20110112_AAAAFT monck_e_Page_16.tif
19f41780192fc01665ef133c591d0c89
92c90cfc73c3c733542d4b2563d718caa5503584
6366 F20110112_AAAAAW monck_e_Page_57thm.jpg
06474267d4d238bb007ca1ccfbba9596
8ee7b968118553d1b90c3252d5d455d901486eb0
6249 F20110112_AAAAUM monck_e_Page_62thm.jpg
375721e1febe680d6868403e416db7db
90ccbc6daa742210f69a54701d851fce36c6f9d8
62865 F20110112_AAAAPP monck_e_Page_15f.jp2
651ca4ba1cfa388175faadd16f42f8fa
a61334a1cfcb37777db00fc201fd32952774410d
F20110112_AAAAFU monck_e_Page_17.tif
f3f5ad72dc7a733dd818ae1b50f791f1
4688fbda8b6c363b3332074278da4e91fa73311b
9844 F20110112_AAAAAX monck_e_Page_74.QC.jpg
0f91db78927a43bdcb3de6bba74b1e33
d52e1b678782fc29d4fa3eacbdeafc6c0e781cc3
24503 F20110112_AAAAKR monck_e_Page_71.pro
de41c772262023bef250c643d755afa3
30c471c1d3df4a9804b3e30ab43d741d1b785b7f
26796 F20110112_AAAAUN monck_e_Page_62.QC.jpg
e5cf2e898c261c70e1fcb939bfadd67d
fb65d09fdb933958442d199feccad78a3d878dfa
6206 F20110112_AAAAPQ monck_e_Page_16thm.jpg
a0a60a11c615e0fbeb92f8b886f1cac9
374478b3408ce91233742e396f8882105422ff37
F20110112_AAAAFV monck_e_Page_18.tif
35cb04c5a9c9ba081c78e30e3b350e31
6840a3a628bc8b7e7ac62ee7293141542e839fb6
2028 F20110112_AAAAAY monck_e_Page_39.txt
dc5f56596730d83f6eda595efd35d5d2
6a45a634bb4b7ed990d7826447dcaf234b89ed72
46876 F20110112_AAAAKS monck_e_Page_72.pro
87222dbd5a63cc1b56d9e6a76d3a2360
fdf64b5f92eb26627727dbb557a6711472fa08e1
65022 F20110112_AAAAUO monck_e_Page_62f.jp2
a48e01934cfc6fcdccc7a39422c4e737
c03bcc9aee393fa85d4e5e4e67cbd12b5394d65d
25564 F20110112_AAAAPR monck_e_Page_16.QC.jpg
838ae840b9bb0981ddd1496e95c2557f
1eb6346e6daedbdda4d650478471cd6f7f4daf2e
F20110112_AAAAFW monck_e_Page_19.tif
752ff39ab8fc545b2e2b2191ce63c00e
080626d6ea7a77d5c2d0cd5a26214872564132f6
66713 F20110112_AAAAAZ monck_e_Page_73f.jp2
bad160ad10d081c49a57a69fa02bc119
15095676c3ae5f39a281572e8471be466c73b138
52227 F20110112_AAAAKT monck_e_Page_73.pro
0c701852748616e3f2c2c78f69fa423c
057bb51cc9486cc2f4c3311bf224b635640ec9a1
6399 F20110112_AAAAUP monck_e_Page_63thm.jpg
d4d5e1a26776741330a7d465fbcca8b2
98d588dd23f6ef2b25ddb863861ad6cb3e75648f
63041 F20110112_AAAAPS monck_e_Page_16f.jp2
c38df3cef3e55b7879258bd2507ed17a
4af37260ee8fddf31fa4c06c0e33663a70d73864
F20110112_AAAAFX monck_e_Page_20.tif
264ad49758e41c43bc92c3dbbe6b6af2
69c1777ed6ebd25f6a48bd7e278b76f083db7c67
58917 F20110112_AAAAKU monck_e_Page_75.pro
f621b47e60aca6afa3c6248d709dc2e1
2a6a26db94ba0751009c33e4f014f250623befb0
64899 F20110112_AAAAUQ monck_e_Page_63f.jp2
e54596a8dc917770902636c18cb10f0c
ee05a92277897337f15313090329ebbda7a51b9d
5124 F20110112_AAAAPT monck_e_Page_17thm.jpg
2d87e91536d634614c6a2314f59aaaa0
df82e473c56cbeed2ffa905111a33e27bfa2c5b3
87810 F20110112_AAAADA monck_e_Page_25.jpg
5ae70e00c2de5b4efa5c980dca386715
3343aa0429322bc58b612002c30054a5534c1001
F20110112_AAAAFY monck_e_Page_23.tif
d985da0151103a3e9d2fce9c215145fe
347534cbde6f8cab01f5242e769680d1145db229
63298 F20110112_AAAAKV monck_e_Page_76.pro
43b9ac982085224dbe00960c421e2753
d23e8dd7972fc066c9f7227328fd3612610cc0be
4689 F20110112_AAAAUR monck_e_Page_64thm.jpg
47fc700a7579862ec9592d6aa2fec1f7
75d8fdde30393826ecc72113a6d5829db5445754
21242 F20110112_AAAAPU monck_e_Page_17.QC.jpg
44cf5803da6d981da1527b5da4336fb7
c256dd735899fad861bb3f231e15c90e82d24631
70959 F20110112_AAAADB monck_e_Page_26.jpg
08190ec50252ab8ef55607f9fe91670b
fce396c64a935165e8f9ac27b502529faea196b3
F20110112_AAAAFZ monck_e_Page_24.tif
a6d0edc6f4c11085435f47a193001f28
540648dd788c1fb18954b3720a00128be61a8a6e
63730 F20110112_AAAAKW monck_e_Page_77.pro
ac25d3e16cc1ae5127098a30d67f955a
2f605ad4bd2e8a4aeb0d104fa8d19545eb7d9f66
18844 F20110112_AAAAUS monck_e_Page_64.QC.jpg
dfd8239594434afdcbd07c88869422c3
19e38711b39ff8754776827c43697a3b30ea375b
52015 F20110112_AAAAPV monck_e_Page_17f.jp2
b2ada9d8e504ba9d795896943af2353e
77f6d60b0ee377ba58c149b75137641f10d7c853
72900 F20110112_AAAADC monck_e_Page_27.jpg
3b13c7598390e04547d01f852ab4105f
a4bd44ab7f805c363fd8b79819dd9a10a299beac
64789 F20110112_AAAAKX monck_e_Page_78.pro
3906a63eee6fe3e3803988873e4df9dd
a32cdb8f828c4bb356a83263b06574dc88319c5d
45081 F20110112_AAAAUT monck_e_Page_64f.jp2
8367217ed590ced298e08f38b18c1df3
cefd919c2f91093df6bb9d65cbd9835d91b6f647
79648 F20110112_AAAADD monck_e_Page_28.jpg
bfcbf12b1b3e22ba259bde2ca9c2c6b1
235700fc31a1885af44f67babfe20d7843a65c95
F20110112_AAAAIA monck_e_Page_78.tif
76a5d08acc5065bf097a7ba77fa217f8
40b516f3f4407e76dda63f78957d405ee3878427
59329 F20110112_AAAAKY monck_e_Page_79.pro
6a4613b873cdd4a87088ecdf2d794185
8a6c2a36624e7362e36b0580d49c1f65fceec8d0
2721 F20110112_AAAAUU monck_e_Page_65thm.jpg
9e9fdb929a070fd8829133eff75c70f7
b556d237e2b04fa88e80b43d3f755774039072c9
5752 F20110112_AAAAPW monck_e_Page_18thm.jpg
3de76b420ca36a0743942d3a98c57b6c
cd76b17af0f8e9f3546b1416234da0ccb158c356
F20110112_AAAAIB monck_e_Page_79.tif
75dba7e26f9ea732be087cdbefa4355d
2d1da557c382d7d5fc547f0393dbab49f73bdbb4
60910 F20110112_AAAAKZ monck_e_Page_80.pro
473baf0cad09883b93d3054377af6f36
fbad4aeaa91e7df1eb25c03e4e7b95610696bcfb
83131 F20110112_AAAADE monck_e_Page_30.jpg
4ad8b736a48ec1d9962af88061e0bd3a
fafcb898da73281b8a6997c056c86d156445672e
10760 F20110112_AAAAUV monck_e_Page_65.QC.jpg
a00f6a26dc988ab697ab289f7cc14609
7aa723987a05c0fe64e427ff99a68a1d2795c082
24075 F20110112_AAAAPX monck_e_Page_18.QC.jpg
8c4ca66422993355dd21fb4a871ff081
fb9daac5f1d82f28d15495aecf04d76d5fec3a05
F20110112_AAAAIC monck_e_Page_80.tif
904d3a1e32d9adb919dbeb15ace57474
f97dd5b186330088d8a1930e4db7d16e7f0f123b
78382 F20110112_AAAADF monck_e_Page_31.jpg
5172187fd9d14aacd2e9b394db284ddc
e115d9ab9284de7d439819029c05e13d7f16c76a
4636 F20110112_AAAAUW monck_e_Page_66thm.jpg
ec6166b306ca1c9ad2d6dd9218653578
3130bd6c230c9801d99e8c0fcdd83ac1a08d4ad2
62022 F20110112_AAAAPY monck_e_Page_18f.jp2
73e72270e315b1e4c1740cb33a19bc8c
743ebdc0178a3e7ac5252b83aaf6df494a6664d3
F20110112_AAAANA monck_e_Page_55.txt
be885d39d2153d38aa3c3f3f58e16c9b
adf604bcebd7bb08118774821d4565a95a1eb254
F20110112_AAAAID monck_e_Page_81.tif
c9ca6d1d2be0c40ca9d4ceb34302da2c
61621a0872794021efb9694c14b88277ff2acdbb
82639 F20110112_AAAADG monck_e_Page_32.jpg
036be5d5824e140d023b5979b167bef1
164148f79126506c56a18900c27ff495989c9eda
15306 F20110112_AAAAUX monck_e_Page_66.QC.jpg
a924efe1a09aabdeb8fe5049679b8b71
517354691318d6bc9c1b8cbf271f48dfb8480446
6739 F20110112_AAAAPZ monck_e_Page_19thm.jpg
f388c30a9b9027fa64aa4389f15e9955
d815bb2df6ff495115ec80949fe37330529621aa
1980 F20110112_AAAANB monck_e_Page_56.txt
41153496e468ae0901f9e3bd4c467373
44a0498b2c15a87b2b84d85d87510dff4e736074
F20110112_AAAAIE monck_e_Page_83.tif
b5ea96a02a0eb6f87deb5edde2bc7bd8
d5e978af4ef6b61b21a08fd503c95c497479ccb6
85336 F20110112_AAAADH monck_e_Page_33.jpg
915e44579e9ef74b135f2bb14365532a
a81646ef98539d33669ee986ea9678a2c8ca41e5
353898 F20110112_AAAAUY monck_e_Page_66f.jp2
6071e22265e07e8ef5e170648ffb8514
cf8f5b2544bee5d9d2740da49cc4953bead3e274
1950 F20110112_AAAANC monck_e_Page_57.txt
c5cb4affbf5b51aef590b1368dbb11b0
efd5c539bc28b32dffaffd5914a1c286f669c4d9
F20110112_AAAAIF monck_e_Page_84.tif
ba90279bd7c08b48ddeab4076bc264b8
869083bea54ac0fe9bb2e3a1517f06b607e2547a
82439 F20110112_AAAADI monck_e_Page_34.jpg
042d6523b0bef0e1f4536930db6ff71c
429bce98370c3dc346bba5746c2ace47dff905dc
12441 F20110112_AAAAUZ monck_e_Page_67.QC.jpg
be02a3ddc9bf5e3deb2976626881717c
58c3c2586187ee3cbaaccb5fdd07ca2a3688a580
64624 F20110112_AAAASA monck_e_Page_37f.jp2
2489eb0b040bb92e23938b48d0017566
912a8cc7fb077e87c32f41e5efb5383f1c525544
F20110112_AAAAND monck_e_Page_58.txt
57acfab20159ab1637fb03805129ae9c
8baaf2ead6cedf5bae800cea3d9150b726fe8d9d
10486 F20110112_AAAAIG monck_e_Page_01.pro
fa87377043129c812f455cb57b801927
0f7c07d4c90581b19bb7acad73e1f913f4fb410f
84179 F20110112_AAAADJ monck_e_Page_35.jpg
c56697b2895c623e96cbeb1dfb3ddb06
760d5cb48347cade2502587b0349c53c4f9bf42c
6439 F20110112_AAAASB monck_e_Page_38thm.jpg
d0beceb6667f9943aae3ddec3c02b269
9e5166ce69bfc8df4a254bd4ecdea6062945ec29
1949 F20110112_AAAANE monck_e_Page_59.txt
a948ff4e1d11465d8f2d57571f70d4ec
3c8c2f202020eb1fb3000ebf2f13798ccbe29061
1172 F20110112_AAAAIH monck_e_Page_02.pro
c8b2c2c69e55c6ce2d48f6f4551bab0f
8d24af99db63aa19cd747b990461853f0282c53d
25379 F20110112_AAAASC monck_e_Page_38.QC.jpg
b879fc9d19aa09526d48b987968dab66
3d0582825836e1d33d1952615333d10ec7fe1664
1840 F20110112_AAAANF monck_e_Page_60.txt
fa96f3f30541a96c3be0113c5ce3f1b9
ad62f7770f5e5e18f0bcbf85b05be8d66787a8fc
3705 F20110112_AAAAII monck_e_Page_03.pro
72a5ce2166d77b4efb2608585717a9d7
4f71fe2d382249549406b01bb874838cf178bece
81369 F20110112_AAAADK monck_e_Page_36.jpg
a1938896119c719ae830e4bb7e479e44
be63b0e59c48f386a66cdf36c80d5e6568d3a8c1
62764 F20110112_AAAASD monck_e_Page_38f.jp2
6281d48cf2f09d6ed4ac642fc55369e7
eced74f378d3765037d775ac8acd42e0a49b4063
1940 F20110112_AAAANG monck_e_Page_61.txt
59366dd63e34b09e0706f25385313430
610a1d4a74a7e2d33cf4ac2a7e454ab491e51eff
24645 F20110112_AAAAIJ monck_e_Page_04.pro
e8c0fde3ea7644881baac813d9d4593a
800bf3f2af844bb061f618dce56473f828f0a52b
83790 F20110112_AAAADL monck_e_Page_37.jpg
4909351f436d95736a61cf198c3b8a2c
a9a6c07212827d549a6b1dba87eaceebf63778e9
5793 F20110112_AAAASE monck_e_Page_39thm.jpg
448594413b11318010be3410fac95cdb
f0f15826d38816e9952c6a05b51d1b17b09c5278
2026 F20110112_AAAANH monck_e_Page_62.txt
39015892c1896a567160c93eb965a9bb
7e22606febe7981cdce0313f2c0e6af547d7fcae
75686 F20110112_AAAAIK monck_e_Page_05.pro
4aa7fbbc264bfec8ff4d2150b53c2144
0385b8ab2db9e72ab2b397128eb1e5a4f6804b3b
81612 F20110112_AAAADM monck_e_Page_38.jpg
1cb927d5ae7b2b3a3e5700ac56822a3a
40404e48be03a41212648aced394d33eabfca04c
23347 F20110112_AAAASF monck_e_Page_39.QC.jpg
42e80ff272222746b44e2d7ff986294d
6345a700d6290fa2c0e13c1e6f2f7a4194790f64
1999 F20110112_AAAANI monck_e_Page_63.txt
4fcab7aa7f57de98bb1a2ea7380652f5
f6e50786a1f5c79749e6b2673af81f9dac13465d
31419 F20110112_AAAAIL monck_e_Page_06.pro
5a151daad9e618bf091c061b224d48c6
7a5064d13d30b9f9dec150743998ec5a19dc2985
44889 F20110112_AAAADN monck_e_Page_40.jpg
476f9761468422ed854567dd8c5db0c4
452ff36f82eec066f07acc85be19907a2b582b4a
467578 F20110112_AAAASG monck_e_Page_39f.jp2
900b82f8573458a53b55a572d668cb35
08bd93a0d051be6c16102f8d15fc82ae0d721a7f
1856 F20110112_AAAANJ monck_e_Page_65.txt
1d5c11afc42583ff5fc90e5befddb8fa
8cdee257b841a41553f855951c2f1b42a93d26f4
12882 F20110112_AAAAIM monck_e_Page_07.pro
2334a9c0442444c0064c72d45a890e15
668c7fd224caa1e9368c743649802fec46dc2cc6
35873 F20110112_AAAADO monck_e_Page_41.jpg
6dd71efa57061f5f78565e89fedd420d
11499803e270e8e87410626d855469bab1e41734
3833 F20110112_AAAASH monck_e_Page_40thm.jpg
b0776a2a5a5a4a436d96f1d8deee9443
05cbf4636e829c3f37532eefedbbefb4de73f23d
846 F20110112_AAAANK monck_e_Page_66.txt
3697228cf3a32728626e2c498ec7f866
0f10ebb6a61efc129f1f96f3426b4507eef43652
30794 F20110112_AAAAIN monck_e_Page_08.pro
ffd9885a61ad6bba19846c1ea5eef064
aae59e923420e87480a8398d4c6ba2d4df467c91
48948 F20110112_AAAADP monck_e_Page_42.jpg
e0773a3f12a60af3b4499bba962fb836
5cc4d12558e9948b604b79ba201acf03655ba7d3
14125 F20110112_AAAASI monck_e_Page_40.QC.jpg
45f5b91fc22bd37830abf4ee728ef977
6e1d3c46640bc20e8991f37d342216698f8bb0d5
769 F20110112_AAAANL monck_e_Page_67.txt
7378cb2f53b26c3ea8ff0507a4fae1ac
471bb647f0016b77a8287f073b9c113de1e97647
41611 F20110112_AAAAIO monck_e_Page_09.pro
f383fcc7aa3b68a6d0ef8909b92e1268
dc89928c94baf5a86764c756457bd319999bc7e2
33056 F20110112_AAAADQ monck_e_Page_44.jpg
c71e51aecc75f4770476a514484980af
413c7ed98b70c2c210b66d3dc5949d4f6d890683
467611 F20110112_AAAASJ monck_e_Page_40f.jp2
3c41ff7300da98e43acb149e58548422
bfcb59e3fba9254482656ff53f215f41baf33260
847 F20110112_AAAANM monck_e_Page_68.txt
f9dfa1cf84223f46a976ff9d3c3915a0
095e1e77777fd83625e848833fc3e02904fdd1e4
80646 F20110112_AAAADR monck_e_Page_45.jpg
84953733a7c14c55a5ce73b03525df4c
9f65c773c08ab51de96a39e73c42dce6e0116616
11722 F20110112_AAAASK monck_e_Page_41.QC.jpg
ff55be3c91a529634e4c3f52fbe62950
d01cc24714a8439122862b12f02b3c5092f6bc0a
45929 F20110112_AAAAIP monck_e_Page_10.pro
5bfc32f79cac1ad01599f2162e6ed7fa
32f3025c87f7674f642560cd4e6c44041a6ba797
80868 F20110112_AAAADS monck_e_Page_46.jpg
5c58942a84b8689d52896ea0409436e7
4b3cc9bd6de97126cc0870e201522fe61a79936f
415 F20110112_AAAANN monck_e_Page_69.txt
76a4c8623fcf21704f98e6234de2a294
133120d2f9a5430cd147c0416ae24de25a73db04
130525 F20110112_AAAASL monck_e_Page_41f.jp2
8c893d6732ffc2bb17a515defa01098a
a32f8ff632e38256d0454933a9759a11f3b66862
44014 F20110112_AAAAIQ monck_e_Page_11.pro
73753958a0721b47ec9aefe5394aab33
9240d428700f4e2f3d97511eb63bbcc58c5048a0
85551 F20110112_AAAADT monck_e_Page_47.jpg
e2aab261fdec5258468e81a52f91e943
4f9a38ee4cb1f582d1246d305869773262925bb6
899 F20110112_AAAANO monck_e_Page_70.txt
94d604eb39e4f575434e96726006ff2c
0d0b95a56a637b572d2b43e1898ea475ec1cb84b
5403 F20110112_AAAASM monck_e_Page_42thm.jpg
5ea000b57f64f2145e479b127371e9b9
bcfa468da87d7c252b5a400c584c60c7f128f66c
51869 F20110112_AAAAIR monck_e_Page_12.pro
9a8f7723c7672390537f2e946ff24bad
21b3dc4ba71aa430a44e79e7390801ad96aa3ace
51414 F20110112_AAAADU monck_e_Page_48.jpg
f198f242723200704870f701d049027d
f616c1e95867b36c4ccd1f05f55d6b03fac4d0c8
984 F20110112_AAAANP monck_e_Page_71.txt
315b5231615a66ad9266872c804060ca
b990d9d53404d59652358fa8b0ae53cf9a9dccab
17547 F20110112_AAAASN monck_e_Page_42.QC.jpg
8bffeeede619c3ae068fe0be5a8f73bc
5786a6415ce13300421989fb6827fed867e5a5b2
53052 F20110112_AAAAIS monck_e_Page_13.pro
03aa881fac11f6a335d6df4e15a4541d
ea0ec555e9fa8347f765b4cf6e47b2097484c210
78041 F20110112_AAAADV monck_e_Page_49.jpg
c209e8ae59750244ce03fb60a26b90ce
95e34f05fc0759e34fb6b0392898aacf6d1353cf
1913 F20110112_AAAANQ monck_e_Page_72.txt
c25927e88b1b4527f2dd9c5e1bce3a3d
78f3c6e5c402ac3135ed1757d2c54666c376aa55
291764 F20110112_AAAASO monck_e_Page_42f.jp2
658ce8ffd72b1fc7f86af243c909e4fb
cb22efc1d76908b7f0616cf1eae701604685ffd6
50894 F20110112_AAAAIT monck_e_Page_14.pro
8fd339a9148184a17f38721e61f7ed31
bca98f6a3f43579ea3eae23095794cabf3f72a18
84434 F20110112_AAAADW monck_e_Page_50.jpg
f3cd2b1f45bc31e94d571c814b00f6e2
7cab14e23c048d7df771ae3b4845dbc5ece6ee35
2049 F20110112_AAAANR monck_e_Page_73.txt
a951dd8bcc150200a56b2a24b76a9d9c
699470be0fb055646b5b01e77738d9d487854f03
5651 F20110112_AAAASP monck_e_Page_43thm.jpg
652b261fd010c95ab758ab63e380e509
b581f374e42a8d4a292edfa1fd6ed409e354b655
48186 F20110112_AAAAIU monck_e_Page_15.pro
a54ba9b935e7a456881c898656966528
ff651e92fb192163c9e0377ed24101704ae8048f
67974 F20110112_AAAADX monck_e_Page_51.jpg
87fd47bc0a0e3b75c5d8115769162c67
d334be564225ac44b17adaae528ee381ee6d6f63
683 F20110112_AAAANS monck_e_Page_74.txt
fef3fb9af7e4c7c84fd608cbbd664cfc
4c63ae3aa878972eba68f97f53f3e257a60e2064
264959 F20110112_AAAASQ monck_e_Page_43f.jp2
77652a0261aa931871cb198d0ddde705
25a6b0fad26e7c846081d04261b8146501cdb09e
49530 F20110112_AAAAIV monck_e_Page_16.pro
7657537062f9380a49e0e0274011e954
817682bea1f03a03761096274aff9c2c9c4e9e5e
63062 F20110112_AAAABA monck_e_Page_45f.jp2
95268f7a6257f6fcefeb6ab6589a2a95
a30f8790b801c8d941a391050137532f63039b59
77173 F20110112_AAAADY monck_e_Page_52.jpg
49577799f871c54176e5a0e223faa478
fa92b1dd1a91dd5216562e45338d1c16706d6ea6
2282 F20110112_AAAANT monck_e_Page_75.txt
7523c2fad7c437916f9201050dae2629
88b3ad9d92274ddca4dc434e2533cb59da2bea7c
10524 F20110112_AAAASR monck_e_Page_44.QC.jpg
695182e4212cc51c31f031d619bef04c
7dc2b5b2b2c32c11dec6ca922813d1f63f7dd179
40361 F20110112_AAAAIW monck_e_Page_17.pro
e07cae2b2b01a1d1760dbfdd5732b575
d4a7bdc279bc617d5cf7704ae3adc71738f37f28
24455 F20110112_AAAABB monck_e_Page_72.QC.jpg
5701a0dd3a5c010bbe6f84c735756cdc
d3ac84634e741bd0c30e43232595f59ac01e1684
86991 F20110112_AAAADZ monck_e_Page_53.jpg
65cc8fbaf8c10321ee66e43d3ac3a6f4
09f15329257977035875de487f2829b8375c368d
348267 F20110112_AAAASS monck_e_Page_44f.jp2
89050d33b02ef1d8791772eb99b36b47
7196c13becb232ae3967e430675dbcebea3eec4a
47253 F20110112_AAAAIX monck_e_Page_18.pro
4c9dce83ec9f4524921c6d055cf4fc19
baccb4e8cf06996d4eaf8352a476bb153c6aa260
6184 F20110112_AAAABC monck_e_Page_36thm.jpg
88a72c7ff0972b032540e69c968614b6
81d126ddfe55ef70116b39d6aca60e03582c3a70
2449 F20110112_AAAANU monck_e_Page_76.txt
28bc792c18c5fdbda8ed958ca86ebcbd
d15dddca201bd92c12791e567d17d0b306d034cb
5955 F20110112_AAAAST monck_e_Page_45thm.jpg
12f6147d981bbee0fcb073c4521cb01c
6a1e26d40b4eabc348d2b99856c0ddd9c0818cda
50833 F20110112_AAAAIY monck_e_Page_21.pro
2f873be3a5d58b76d5be5a503ef1cc6e
32d7a3579188ea15f02cf76a47d9e954b6efceb2
4125 F20110112_AAAABD monck_e_Page_71thm.jpg
1c49c9c893b4d53ffa1ffacd35de0a5d
10c7275745de89468c1deda2140053f54e754a0f
2507 F20110112_AAAANV monck_e_Page_78.txt
35374d937ad15cd822ef8fa149bcd5a4
433472ff457254b4ef2b189e06ae0fd7af620b5a
F20110112_AAAAGA monck_e_Page_25.tif
8f7362ad3dda725277c1d62a304aa380
233deef4bda5c75a8ba212c9094b453759f91158
25250 F20110112_AAAASU monck_e_Page_45.QC.jpg
1fa4dc2d9be760c554d7c3986c5424f1
78600fa352d5a0dc5511cc008ba2b1aca284302e
50400 F20110112_AAAAIZ monck_e_Page_22.pro
164a1e67fe1132124f9c53413ae2c11b
18a07fcfbc4be6c8b65b6a96ce8058f185cf5d9f
16510 F20110112_AAAABE monck_e_Page_48.QC.jpg
487325f24afdef185b191b7bdbb38721
54e16867d62aa79d86ac08c8e07f6025a602c504
2306 F20110112_AAAANW monck_e_Page_79.txt
8646f1ea6210f30b5b87683e319a26d4
e3d2090ee679141cc110a2e9765ef6a07634ae77
F20110112_AAAAGB monck_e_Page_26.tif
c9c3ff480c3bab77c1c797271b6052a1
5ec097253be8842239394574fc12b3d33a770467
6512 F20110112_AAAASV monck_e_Page_46thm.jpg
709fc090cf690612f820ca6d73a8cac4
01a5073404ef953ebba5a61b94ff0597d2a50c1c
3486 F20110112_AAAABF monck_e_Page_67thm.jpg
54dac51b4bb06f2a61fd0377c0099c84
c1a7f79303710fbe2aff556ce87084927e271ca7
2366 F20110112_AAAANX monck_e_Page_80.txt
63c74d5b3bad450147e67caf393db09d
a4962c0ae9c479b524f611f3c3961386138eecfa
F20110112_AAAAGC monck_e_Page_27.tif
ca4509c36bce143164463e8b50dcbdd3
af962a8d7996ac6e6b557c0a2767d0cfe933d82f
25721 F20110112_AAAASW monck_e_Page_46.QC.jpg
23095652a31ed5dd46b943a58979a192
d7505e953c16be31a89d0288a289d92cc68cc0ce
49446 F20110112_AAAABG monck_e_Page_57.pro
205699be55c943d13a9c279bc54091f1
64930338a3c6331d8c26a9f1f16a59b61d816b42
64172 F20110112_AAAALA monck_e_Page_82.pro
bfdbd4a3555c5e0931c9bc799c791e8f
73a0ff3a670fd761b6f0a5bf3e5a1e21af0dc866
2653 F20110112_AAAANY monck_e_Page_81.txt
2bfb0fb16ef6a5bd8fa0959acf4e2196
8c2d728204a0d97516a647db980d8cde70d9e88c
F20110112_AAAAGD monck_e_Page_28.tif
a57d54393b02c50cdd1768d13509aa93
dac16f932e0b69a4f259a56c3a9a43554033a701
63419 F20110112_AAAASX monck_e_Page_46f.jp2
0d693504de3d24684e1d2527ab434871
f88ee3f7f40c53a164b678e472bbf1776c9f5665
F20110112_AAAABH monck_e_Page_21.tif
a4164be4632a885d7ad15bedad9f316c
9dd19f15efbdd6172b95a21161180318fdeef000
21061 F20110112_AAAALB monck_e_Page_83.pro
f64abdf2895b612428a7289835bbd19c
9658cb3e51064a3c0373e5f9b876a2a03b4169e4
2484 F20110112_AAAANZ monck_e_Page_82.txt
c6de897b3260a6bdcc61347a48117ca8
79ebc7c2bdab03f2a9089e4ceaa057af8a057bf3
F20110112_AAAAGE monck_e_Page_29.tif
54529d8078d7a2145fa21f63576cb739
2a6db8b9573a7008531bbdb62d56109edb07da00
6622 F20110112_AAAASY monck_e_Page_47thm.jpg
b98a0f24f2d52e6822561945bf6828d6
8932c783e25c4ef27df27a8373512fad84c8a623
32256 F20110112_AAAALC monck_e_Page_84.pro
a5eeb1dab677ac8e2635595d4fbfd112
3c9fcebed22d1cafd416c99e8f179a374ed2c0b2
F20110112_AAAAGF monck_e_Page_30.tif
c8eebf6469421f821d719b6d143c41be
e3445db843d252c573b9d4a84f5ca68cd341ee32
28266 F20110112_AAAAQA monck_e_Page_19.QC.jpg
7665803d3ee7fcd09f38c2343e719c94
342c76e1f6897343922d23b5463a9248ffac8e1d
6047 F20110112_AAAABI monck_e_Page_07.QC.jpg
11abad9482f33cea2387b86443a60002
ccf38d3490bf0521ab815a300c38d62304626a1f
550 F20110112_AAAALD monck_e_Page_01.txt
713d1ba8d33c5043f6e273735734bfd5
c009c4b99f6aff6e17e5aa7ee5ce6df59877af0d
F20110112_AAAAGG monck_e_Page_31.tif
710b6fb640b82bac7920c6e6a3046138
a31e8c81154d4c4dd7e34e54832f8106653e37d9
27161 F20110112_AAAASZ monck_e_Page_47.QC.jpg
3268e2729752acccdc78cc1c380f1160
4087ed89835d4d0620dfde2b51ba7ecc92d7ccb9
71009 F20110112_AAAAQB monck_e_Page_19f.jp2
94aecdc278e06db91d823c6ba024fe9e
6a26ab72e912910ec76c2c91824828beef76cb9a
1975 F20110112_AAAABJ monck_e_Page_46.txt
fb4d8872f924c251aab9604da2a93083
ef0a040e9512163576441f350fc88a4598d258f7
112 F20110112_AAAALE monck_e_Page_02.txt
00b72429e76f89bc1a0f7925838e4c2a
04e753e78728ae6c75955a7efcd828b0bb1fa9c4
F20110112_AAAAGH monck_e_Page_32.tif
cf981fe2e31422bcb006c45e0f7386aa
fcd30892adf345202b888a87031660eba39e9bc6
F20110112_AAAAQC monck_e_Page_20thm.jpg
973554b48edf400bd99564fc22a68b38
b5362941d6baf30f772770d5a624882819b3a3be
5454 F20110112_AAAABK monck_e_Page_51thm.jpg
744730797ba03780998516ba14297ce8
e2d128c81d193580046114868f37d3696dbfa600
216 F20110112_AAAALF monck_e_Page_03.txt
4f1e79a2e7cade78b961fb6692cf1db9
77ecb3733a39d2c54f46b8c5bd72a501e43f2692
F20110112_AAAAGI monck_e_Page_33.tif
87be5ec4d89e04b40585aea55aee0a78
3146af80b8fa56bee2fead890755680cbb27a833
262880 F20110112_AAAAVA monck_e_Page_67f.jp2
759607bc276c5588b2c2ea80d9c83ba9
ceb7e95bd546b637c921094db2319eef61e64dc6
26295 F20110112_AAAAQD monck_e_Page_20.QC.jpg
17d9e5584d3b1fb51fdba4dc19a5a7ce
d233e22b13517a92ed908ad25d6ef0abb26b07c5
136599 F20110112_AAAABL monck_e_Page_69f.jp2
43dd3a9d8dfc9bac39c21e0956e4a80f
3b9aee4dbc941bbbc32c4e0fb4c46f168611f099
1039 F20110112_AAAALG monck_e_Page_04.txt
f9de11f41e96830ef66aafd6fd9d5fc6
673bfd8b5e12063647eaba5e5797bcd3e5b0af80
F20110112_AAAAGJ monck_e_Page_34.tif
c9a67f7d2d227a761e71d7f5faed0ec8
bc7139a478de8e2056f075c3fcd01455aa2e7640
4383 F20110112_AAAAVB monck_e_Page_68thm.jpg
24dddbf44012e7ee8be41c12e47d2a05
33a167f82021a8958bdd421de29184ee04666930
64337 F20110112_AAAAQE monck_e_Page_20f.jp2
87b0b01390589cca8c2ff0938edbe296
26e7c3d9847b088fdae9586578e428b7ee680184
6568 F20110112_AAAABM monck_e_Page_50thm.jpg
8e2a417bbe2b2fab6d303c70e8aab19c
8eef920a476b2e2762230959bd1305dc5360363f
3107 F20110112_AAAALH monck_e_Page_05.txt
7feb9a0966e30d373263c5a9cb94346a
64245ca93946f91f3ed8f2e502c1f366293cdaae
F20110112_AAAAGK monck_e_Page_35.tif
a7a51160cd52ff2cd38b3a0ba059d6dd
22d240676e97b42bdc08af43ca7f1c77c70574b8
16023 F20110112_AAAAVC monck_e_Page_68.QC.jpg
435520a8218e8c074b29c3f22b627b1b
d7d7c4127c5d75edf2fcacfdcf86719aae5e3328
6418 F20110112_AAAAQF monck_e_Page_21thm.jpg
dfd3d5b40777263e4b36b84a434b9de7
5d4f406209c8f2147c9b44ad77a142f14462ccda
67385 F20110112_AAAABN monck_e_Page_43.jpg
acb1ab873dde10dcc79f1bcd02f53f3a
5794d6cd202d0b2dcc64809cb01a4c7c19a06321
1285 F20110112_AAAALI monck_e_Page_06.txt
cbe3f8e05deb9ba43096153f8ff1ab7f
2bb719372ee5d09ecc7714a8640ffc76e831c758
F20110112_AAAAGL monck_e_Page_36.tif
94900838572b736b99dbb59732d9060f
382b6b9e1fd0cc52908ca0b8b4b18b7370c4d6a1
263051 F20110112_AAAAVD monck_e_Page_68f.jp2
12fe1c6f5fd83b1bd7a350af4e5d9286
e7735c0fe3467afab436d4a93ab686ea17245897
F20110112_AAAAQG monck_e_Page_21.QC.jpg
f17440143db52150637d01c56cc6ec79
2185c286d64ff630f7369a5c2087989207670979
F20110112_AAAABO monck_e_Page_76.tif
83ff4a37369fc976641c9a250f38c24f
95ebb7f2878b0c04e3b201afa2b66bf44ce277c9
556 F20110112_AAAALJ monck_e_Page_07.txt
7b9fcc2f0dd774c7594f54cdae2836e4
fcb56d3c8b488cdc34cc203e58831f9402d0574a
F20110112_AAAAGM monck_e_Page_37.tif
e1f1455d5819d1342847d6a410ea2072
64164386cab545a404b956f9e20b7c48ed22d56d
2636 F20110112_AAAAVE monck_e_Page_69thm.jpg
d2b10760e0f7b2e46fcdfe15251dc119
01cfa9e2e806c253804a25e882da155f7d5f4873
64787 F20110112_AAAAQH monck_e_Page_21f.jp2
233bc0aebed0ad1072d08f22e59a301e
7644474fab896b8432bcc8c16d2931aeab2e28cf
3260 F20110112_AAAABP monck_e_Page_44thm.jpg
cf68dd0aa785fafa732284a3abddd52a
02e15ea0484948362e902822cdb0c913784359be
1264 F20110112_AAAALK monck_e_Page_08.txt
4077c18d2aa1557476f12dad26a933fc
d6eaa81d0e80155a6ad9d7424e6c29ff88bc9a4d
7881 F20110112_AAAAVF monck_e_Page_69.QC.jpg
dba572416a19c1177fef00b7b4cad87b
90f8c523dda47145bcaf126b9d3e8631bc308204
6461 F20110112_AAAAQI monck_e_Page_22thm.jpg
0c508d488f1ebf7759a02fe053d0ed98
55a2bf276aabdbaa64289cb7f43035ee92338104
81878 F20110112_AAAABQ monck_e_Page_29.jpg
1df5b5c94fa4048593d0122a10f63c7f
0bde87a1275539567795e62405197f98cb4ac809
1818 F20110112_AAAALL monck_e_Page_09.txt
c34f6384b5d4c4e13d12cc472794f522
ef91108df7ee5b8b19cfa915260564a4e71bdb71
F20110112_AAAAGN monck_e_Page_38.tif
4ca87848a54b6dfcda29a053b4eb3e05
9ef98c28b3e889450680ef9acd3f2fc3cbc040e9
6282 F20110112_AAAAVG monck_e_Page_70thm.jpg
4dfc6aee08f49a834bd2c1ce29deb382
439008a2db927cf46c34ef8da9d13c341479be0a
27052 F20110112_AAAAQJ monck_e_Page_22.QC.jpg
284dbd7a3d82401096fca5f8dc38842f
a98a7e3cf1dde3f9eee4f5d68fa2b00742783a6d
56322 F20110112_AAAABR monck_e_Page_19.pro
4e0a4ae90900169a6720d9896740e10b
b552ab212fe51564ef654c8de226ac445b7f1b81
1824 F20110112_AAAALM monck_e_Page_10.txt
6b527223fcdda24cf6692573f73ce4c4
ebe69076b4095cabe7e66351da097122c6ab9ecd
F20110112_AAAAGO monck_e_Page_39.tif
642b5544df70489954e2ac3be84800b2
a3ee581d12a162a3239454cd2235f0415ed31923
21580 F20110112_AAAAVH monck_e_Page_70.QC.jpg
4e35c8115f0c250c23d2d03da3cd46a5
7939e736d583d4154cda339cedc305682255dc38
66396 F20110112_AAAAQK monck_e_Page_22f.jp2
e20177fdaedb939363054b5bc6463253
99ec5909874eeeef3d6e94cca9921eb8e7e17fbb
27296 F20110112_AAAABS monck_e_Page_79.QC.jpg
00e7f184d432dfaf7bec84feb5cd9cc3
2463050d789df69f8cb7ae4e6c7994a963431f3f
1809 F20110112_AAAALN monck_e_Page_11.txt
eff10e56d18b1786785be507b5a8033e
8bcdc2497a12dc6a71dd3ebb20a69b7ef7e3989b
11233804 F20110112_AAAAGP monck_e_Page_40.tif
3b45e2bd46ff22c17d4d85aaa2b5c26d
41dbad11add58dba9747e162aa94f6866103d646
515457 F20110112_AAAAVI monck_e_Page_70f.jp2
e8139e9e8e3ed79e0ce7ee51cfccec64
093b427223728720f349ac09714e8710be55a9bc
6325 F20110112_AAAAQL monck_e_Page_23thm.jpg
6cadc7f8acc23086e36b3d4316ba600a
eeb38fc59175df091863527b10c811e28b957ddf
1969 F20110112_AAAABT monck_e_Page_30.txt
83f16e470ea4f597234b12870a5f8ccf
068d8fbb953ad08e8c736dd5b8ee922bf4edda58
2058 F20110112_AAAALO monck_e_Page_12.txt
90fd32fec29a743b54660f61591c3b40
1bcf0aacd8574fb107a8d8b97925853a1ae258ca
4601548 F20110112_AAAAGQ monck_e_Page_41.tif
37b8c054af244a2af75a44bc45219c2c
ca5c13d535483edd17a50a9a39c1705590e81772
15970 F20110112_AAAAVJ monck_e_Page_71.QC.jpg
9ba14f286092ee0de5204c14a16027f5
79570d10206b92d6d43bc3e1f9ccc1379da09d5e
25886 F20110112_AAAAQM monck_e_Page_23.QC.jpg
50760e9cd7a07a80771a6baa98b1484a
14359daf5b88839079c97734babb1b2794dcee15
6473 F20110112_AAAABU monck_e_Page_35thm.jpg
7f70e6c36861efd3203a2b54ea7b9d63
f54556c75454ef92b83f2a4780588fde9397d057
2083 F20110112_AAAALP monck_e_Page_13.txt
762f5b4382cfb2e43f80fcba785d474e
dab648bb9ba834e4787fa51274b0e91abc53d84e
F20110112_AAAAGR monck_e_Page_42.tif
9061ab3ab2b3cb2d8682e5eeb6a2edac
a01ef5e4b7595f125010151c81dc1829fae47c01
262991 F20110112_AAAAVK monck_e_Page_71f.jp2
aab44184fb1bbec64e3406e5420ee50b
3712de56a1a03cec0f6fea0aa932af938fc95002
63750 F20110112_AAAAQN monck_e_Page_23f.jp2
c87897def07a286959414bbf8cfaa8f6
4c51002444ea163b32c4bf8c2b14a69284214a20
F20110112_AAAABV monck_e_Page_82.tif
ed4fbc89e399b6a49b0792e01d9de0cf
e064453ccb6265e813a67f5ea758bb9f68056856
2005 F20110112_AAAALQ monck_e_Page_14.txt
d3c261591d5782d50cebc580c7b92386
0d836d0c744f7c126015ba822218aca9e2ed0bb6
6364002 F20110112_AAAAGS monck_e_Page_43.tif
7651fd48e9ba010ffacf0e4eb732b763
fa2c2152a1f2d67101ab0ef52d1ab1be68743b8c
5748 F20110112_AAAAVL monck_e_Page_72thm.jpg
244e61f76f57c0c585fcbf5dd988383d
ca2bc73a01e49aabaa3c5e5b2e512c905e2a8f22
6350 F20110112_AAAAQO monck_e_Page_24thm.jpg
e3988886b169e074848c59dfa25f5a04
c79b33d65aabd0646f46a14e11f09231b5c3525b
871 F20110112_AAAABW monck_e_Page_03thm.jpg
1756ed220b55c8e08ca3b6948c70dfbe
8243c5da8e166b40890e46c567b1b77c9909259e
1944 F20110112_AAAALR monck_e_Page_15.txt
3e8b142ae6e9babc43ef27b9efcec27e
fb191aac8ea2030abeac8bf232cd6cb881e3e56a
F20110112_AAAAGT monck_e_Page_44.tif
979d9c77421d050a880fa07fadcc8ecc
785ed5f00cbd702cc19ee066f8ba5f432d8f0c13
60599 F20110112_AAAAVM monck_e_Page_72f.jp2
1e1d19e6a0ea09372ba5e5ab55e9c909
7e9db45afaa7cf66046e11fc2373ce90f674bf57
25907 F20110112_AAAAQP monck_e_Page_24.QC.jpg
479f6225684e3f2b48abfb8bcdfb1e46
2d07e315b7740c773f4bf2fd0ea8e7861561ab14
467605 F20110112_AAAABX monck_e_Page_05f.jp2
22fe351ff9dac613d6c3db440b94c58f
19061e20f880216a9a3fe03d1093373499982fd1
F20110112_AAAAGU monck_e_Page_45.tif
641dc30fec354dc305829b3f97708593
a6404bf9f01c7ea629079070a5119f66ee576e1d
6559 F20110112_AAAAVN monck_e_Page_73thm.jpg
46e9c6be3a637e21e641b124e1a233de
f3a74858997a3f965cfe340dc3f9c00900dde350
64409 F20110112_AAAAQQ monck_e_Page_24f.jp2
a20a77bb0746450c71028cfd6f541d0a
2b6a16cafadacbad6d9d377c723493317d2988a3
45903 F20110112_AAAABY monck_e_Page_65f.jp2
389d26c7eda339228ef906525c5848a0
e1daa1097cee03c19c2dc0d25acb5a291e68711f
1961 F20110112_AAAALS monck_e_Page_16.txt
724cf17dbe81990842fb432f2bdec413
fd2d6414a6941d2740a596be347f63176b5fda04
F20110112_AAAAGV monck_e_Page_46.tif
36a2b77054eea0b4e5d2aa4ae0161484
5b1f094beabba197398f9dd5598943b9f75afdb6
26988 F20110112_AAAAVO monck_e_Page_73.QC.jpg
f4adf8c5deddab131fc5d02b1745132c
0e09ffd1e99163b39e053cfa46339eac7ade0b32
6571 F20110112_AAAAQR monck_e_Page_25thm.jpg
98ca4f0ba2b3ac46a959d1b5971f3d3c
bbbf8c112855a920861a3d50f01c1a741abd296e
16127 F20110112_AAAABZ monck_e_Page_74.pro
95ddbacdb3ddc8f2032650109ef9e6ba
ad56a5294e7850c0c770df7eac9f5cf35e6884a7
1616 F20110112_AAAALT monck_e_Page_17.txt
cf44df9b49006ae4401d70c86dd336a4
4819c3a21467edce910e1b06c839866d7f83b37c
F20110112_AAAAGW monck_e_Page_47.tif
e7c05c12ce3a8a2bf51c1190ed16346f
c0c3be1b806d183ec67093c0f79af6b843a6d4ba
2350 F20110112_AAAAVP monck_e_Page_74thm.jpg
af38b9e96f8e82705049bf6961cee48e
7351b652b4094562b2594a2822203d5c3dcb0aa7
28100 F20110112_AAAAQS monck_e_Page_25.QC.jpg
8c6b669bb9276cac159e3b65ae914c0e
90bc9e3ea5796c4b43ae2196c3a2bf92e16264ef
3252 F20110112_AAAALU monck_e_Page_18.txt
eea8beaedfe71a8de05a4db2a1b06ed9
2b48070cf3d23ab2236b01f0e7b7059680a23522
F20110112_AAAAGX monck_e_Page_48.tif
98cb312671b945fd0739d3c31ebf3173
8c29c8d905c4aef89c2ef379400ae202085c0256
22529 F20110112_AAAAVQ monck_e_Page_74f.jp2
88d1e06d1d31e4c8c6b0c37ee712f2e5
7114f3a51ec06cc4df3c5d4a4f53cd13e4094c5d
68586 F20110112_AAAAQT monck_e_Page_25f.jp2
42a5b2f46834f5076d4c8490b5d20301
0727b2c006d708f689fbf5cf45aa78a666ff7d49
2251 F20110112_AAAALV monck_e_Page_19.txt
dff6b85b168fe4d00f3742e70f03cf92
7fc00ff0e49e935c1a9434e348bda8524f1c1be5
73104 F20110112_AAAAEA monck_e_Page_54.jpg
8d298567ba365c1884e2de294dc00c53
0305c786c638069a763600bd15c28509eb61f89e
F20110112_AAAAGY monck_e_Page_49.tif
0956d92b509446c2481f143fe67f91d9
a1c532f20843c9252674f483d93af375048f53bc
5732 F20110112_AAAAVR monck_e_Page_75thm.jpg
75d29530fd0ab456a88df40689f2261c
f35a2d9c07cd415609affa087805f910d4e8c727
5567 F20110112_AAAAQU monck_e_Page_26thm.jpg
35bb931c17dc72470e8779100db78e05
2f32f1a9c4c3ce155c7962858af8e79d043635cd
1986 F20110112_AAAALW monck_e_Page_20.txt
e8a8a3b6a49352a2ee4200ff24acb317
c2a637afcd6699d4a4db97b264e01c5b513b33b6
83259 F20110112_AAAAEB monck_e_Page_55.jpg
8d3c30d143348fbeb5ad0d237ed15640
fa71626bd3394b862ef2da01230141beb7a5e979
F20110112_AAAAGZ monck_e_Page_50.tif
b742a463a6b456839800fd2ea9a757d1
1acd5b09f8a0e88e3720ca60a4e309f8380b7535
25173 F20110112_AAAAVS monck_e_Page_75.QC.jpg
86b26e826ea7c331da8f630a1e598325
1f92cddbdefd40e678ebd2d07c100e7cdc1384e3
22811 F20110112_AAAAQV monck_e_Page_26.QC.jpg
2fd168e084f502f3e7a94eba3729de92
1253f12ff7b19aeab581d55b2cfcfc20ba70e764
1996 F20110112_AAAALX monck_e_Page_21.txt
07e68c177876b8b56ea19c8e18b4eceb
a5750d8097debe6ab0b46063f7e9e48d42cfddb0
85321 F20110112_AAAAEC monck_e_Page_56.jpg
c6e72877a021e5e38afa6b26a2daa7a1
d2c4841367173ce06c4c1eddf8acc43eb1df1c66
75334 F20110112_AAAAVT monck_e_Page_75f.jp2
4fa02c6e56e44e001aeb3c8ff99ffcd5
b1762538b163cfb60874c4931c579dc5190db2af
55701 F20110112_AAAAQW monck_e_Page_26f.jp2
624d5b2a7fcef5b08d520410baa2043c
ba6a3ab5e6f727dcab56e510c0ceacda8dfaa830
1978 F20110112_AAAALY monck_e_Page_22.txt
dd12340ac26568fa10f22b4da8279d2c
ad381a0f0f49146c8773ad70984b384b8d927123
83720 F20110112_AAAAED monck_e_Page_57.jpg
d4b60bd00a0f0d0e8c44356d3419de8d
27713d086f9d90282cc393554f8d5fade3aba49a
49368 F20110112_AAAAJA monck_e_Page_23.pro
9bfab3e081be77a16355355b3c61a156
c58f37cc86912dbe104f1dd3333aa7c3483bba7d
6700 F20110112_AAAAVU monck_e_Page_76thm.jpg
347c773fab88a878d0c1d7eda5a18eb5
edc8079e851366babf663c327140d41953e365ba
1955 F20110112_AAAALZ monck_e_Page_23.txt
9db6cfafead78f4c7c32b93117e7d19c
dc0573a0b2b0bbee49f40f39b58bd0abde2be78e
85077 F20110112_AAAAEE monck_e_Page_58.jpg
54b77ae0c03e97f44c7c6ab6d6b6b024
a50130a0049f195d829dbc4c36b39494fbf6e78a
49889 F20110112_AAAAJB monck_e_Page_24.pro
3c827cd1d3fa8e0d3f10ed3d65255f29
8b648dd39256dcb8b17ddfcd8413a2e5fc9779eb
29202 F20110112_AAAAVV monck_e_Page_76.QC.jpg
e5792e0231474960ab9e06684747b1b7
29b6646d6110e98eaa5d8846b4c81b8044c44fb7
5494 F20110112_AAAAQX monck_e_Page_27thm.jpg
b625fe593ce5cf0f6914ef2d1aee741f
21a9475ca833e2b7176ff63f20fc0b95e591c4de
83974 F20110112_AAAAEF monck_e_Page_59.jpg
441eb91dd931d6b95886f898c4392c0f
91872aaebc4dd91842fd433b61b2873f4d922c23
53631 F20110112_AAAAJC monck_e_Page_25.pro
1349ab1c74b81b9319246fccc9683333
13ce37fed8f7517a3a016219b927ced9ffdd26ab
82629 F20110112_AAAAVW monck_e_Page_76f.jp2
ebd3e826e84581e84b23e9e9d417cff6
6f57a4c279097764d349b23417af7655e5f24964
23056 F20110112_AAAAQY monck_e_Page_27.QC.jpg
f128a29a92ba18f8cf3274054e8740f6
67e6c2edbf4c05418dd4d492965719cbd873a379
76838 F20110112_AAAAEG monck_e_Page_60.jpg
aa46a1f845f4c778501799e643d39b14
d86f5c8713d5db5ff1f62a5a8bb0bac77248b885
864 F20110112_AAAAOA monck_e_Page_83.txt
64f4a19475d28a1644b746e7223ea82b
05475f570f77950dfe29ff53df4d148cd0adf5eb
43193 F20110112_AAAAJD monck_e_Page_26.pro
1117f16dd8cf955e0f7159e7631872ac
1095a3c4504b59d018487d0c1133026097465975
6522 F20110112_AAAAVX monck_e_Page_77thm.jpg
5e59315427b401f671975b3b259f2c78
fcc85fc38ed7824012831e8ebff7585abdd1af96
57054 F20110112_AAAAQZ monck_e_Page_27f.jp2
2bb637c7a56686ba4e0d251dc135ed2c
a80f76f605219ce60908e7a1aca186b5366d3f27
82510 F20110112_AAAAEH monck_e_Page_61.jpg
c6a0a7ed7f0bce6c7e6e123dfc7c8bc4
8f87b3b1e239b6890f74d404d2adaf5737ad05c2
1346 F20110112_AAAAOB monck_e_Page_84.txt
036600549501df204f36f4e3b3876845
70c4b541e36f94640ddb230c6cba8c9a62f0342c
43486 F20110112_AAAAJE monck_e_Page_27.pro
6e12f83208d63d5eba562e7b19e16635
3f45e29c236ad68bb2a056556bc0e9f3459bd5a8
28694 F20110112_AAAAVY monck_e_Page_77.QC.jpg
6f575dc9266824cead3b59515d3861dd
d49e798fe914fa6c4d49252ab2c9be9a81d0b52b
85365 F20110112_AAAAEI monck_e_Page_62.jpg
7c5a284b53d69c4d5f2854e77c2fed41
e89b70f0f1a1f6c2b7ab5ce94b8ec9a5df876ef6
1922880 F20110112_AAAAOC monck_e.pdf
7b604b8564a28b6048f9e95389303016
d1b3f3b9e30a80843f0e67499bdbed9225e8765f
48548 F20110112_AAAAJF monck_e_Page_28.pro
1db79d57558b337527fc2eeadf8f1bc7
a59772c410b1e7e5513e0d690a48335726583caa
81934 F20110112_AAAAVZ monck_e_Page_77f.jp2
aaeef590f61612926de162806fea1bf4
536ae92e3e0e9cb199358ddb1b186c1c482fe006
66640 F20110112_AAAATA monck_e_Page_47f.jp2
14f07461a94fd30c1a99ce5ce3a340bd
73696233d53c073bbc489858476d5830a6131d31
83180 F20110112_AAAAEJ monck_e_Page_63.jpg
23cfee156117097123334e766db6535f
a18714d59a7b1775a1eb761d20736f34bab2e9b7
F20110112_AAAAOD monck_e_Page_01thm.jpg
2a5634529326e1c1908dfb9f9ad9873e
9d64bca392fb02e1aab15998a3378409a0f1717a
49385 F20110112_AAAAJG monck_e_Page_29.pro
80b4f50651c4ccc3a1c8cb1e87b47920
3709f5bc368d253108867327bb2305b3cf68dd9c



PAGE 1

DEVELOPING A NONINVASIVE METHOD FOR ASSESSING REPRODUCTIVE STATUS AND CHARACTERIZING GENDERSPECIFIC PLASMA PROTEINS IN THE AMERICAN ALLIGATOR ( Alligator mississippiensis ) By EILEEN K. MONCK A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2003

PAGE 2

Copyright 2003 by Eileen K. Monck

PAGE 3

I dedicate this work to my family for thei r love and support, without which I could not have been successful in achieving my goals.

PAGE 4

ACKNOWLEDGMENTS I would like to extend my appreciation to Dr. Timothy Gross and my other committee members, Dr. Maria Seplveda and Dr. Evan Gallagher, for their guidance and encouragement throughout my studies. I would especially like to thank Dr. Seplveda for her tutelage in the preparation of this thesis. Special thanks go to all members of Dr. Grosss lab for making this study a success. I would like to extend my gratitude to Dr. Nancy Denslow and the ICBR Protein Chemistry Core Facility staff at the University of Florida. They provided an accurate and timely analysis of my samples. Also, I would like to acknowledge the agencies responsible for funding this project: National Institute of Environmental Health Sciences Superfund Project #P42 ES07375; Chlorinated Pesticides and Developmental Mortality in Wildlife, and a partial grant from The American Chemical Council to Timothy S. Gross and Christopher J. Borgert. iv

PAGE 5

TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT.......................................................................................................................ix CHAPTER 1 INTRODUCTION........................................................................................................1 Alligator Reproductive Anatomy and Physiology........................................................2 Age/Size of Sexual Maturity and Sex Ratios........................................................2 Reproductive Behavior..........................................................................................3 Reproductive Endocrinology.................................................................................5 Female Reproductive Histology and Anatomy.....................................................6 Vitellogenin as a Reproductive Biomarker of Endocrine Disruption...........................9 Background and Significance.....................................................................................15 Study Objectives.........................................................................................................16 2 ASSESSMENT OF REPRODUCTIVE STATUS IN FEMALE AMERICAN ALLIGATORS...........................................................................................................17 Materials and Methods...............................................................................................19 Study Sites...........................................................................................................19 Animals................................................................................................................20 Plasma Samples...................................................................................................21 Female specific protein determination................................................................21 Circulating Hormone Concentrations..................................................................23 Necropsies...........................................................................................................25 Results.........................................................................................................................26 Discussion...................................................................................................................35 3 IDENTIFICATION AND CHARACTERIZATION OF HIGH MOLECULAR WEIGHT FEMALE SPECIFIC PLASMA PROTEIN BANDS................................39 Materials and Methods...............................................................................................41 Study Sites...........................................................................................................41 v

PAGE 6

Animals................................................................................................................41 Female Specific Protein Determination...............................................................41 Enzyme Digests...................................................................................................44 Anti-Phospho-serine, -tyrosine, -threonine Western Blot Analysis....................46 Alkaline Labile Phospholipid (ALP) Analysis....................................................47 Amino-acid Sequencing......................................................................................48 Results.........................................................................................................................50 Discussion...................................................................................................................52 4 CONCLUSIONS AND FUTURE DIRECTIONS.....................................................62 REFERENCE LIST...........................................................................................................65 BIOGRAPHICAL SKETCH.............................................................................................74 vi

PAGE 7

LIST OF TABLES Table page 1-1 Stages of folliculogenesis.............................................................................................8 2-1 Mean standard error of body measurements...........................................................29 2-2 Mean standard error of the mean for reproductive measurements..........................30 3-1 Amino acid sequence alignment resulting from BLAST search................................55 vii

PAGE 8

LIST OF FIGURES Figure page 2-1 Map of the Oklawaha River Basin, Florida................................................................31 2-2 Map showing location of Rockefeller State Wildlife Refuge.....................................32 2-3 Sodium Dodecal Sulfate Polyacrylamide Gel Electrophoresis Analysis of plasma...33 2-4 Photographic documentation of reproductive tracts...................................................33 2-5 Follicular frequency distribution in right and left ovaries..........................................34 3-1 Sodium Dodecal Sulfate Polyacrylamide Gel Electrophoresis Analysis of plasma...56 3-2 Glycosylation analysis of plasma samples.................................................................57 3-3 Deglycosylation analysis of alligator plasma ............................................................58 3-4 Alkaline Labile Phosphate analysis of plasma proteins.............................................59 3-5 Phospholipase digestion of alligator plasma..............................................................60 3-6 Western blot analysis of phosphorylated proteins in alligator plasma.......................61 viii

PAGE 9

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DEVELOPING A NONINVASIVE METHOD FOR ASSESSING REPRODUCTIVE STATUS AND CHARACTERIZING GENDERSPECIFIC PLASMA PROTEINS IN THE AMERICAN ALLIGATOR (Alligator mississippiensis) By Eileen K. Monck December 2003 Chair: Timothy S. Gross Major Department: Physiological Sciences Organochlorine pesticides (OCPs) in Florida lakes have been associated with decreased egg hatchability and increased developmental mortality in American Alligators (Alligator mississippiensis). Although concentrations of specific OCPs in yolk do not correlate with egg hatchability and hatchling survivability, a complex mixture of OCPs may decrease egg and embryo quality by altering maternal reproductive function. Vitellogenin (Vtg), a follicular precursor protein has long been described as a biomarker of endocrine disruption in other oviparous species. However, there is no documentation in the literature of a definitive test for identifying and measuring Vtg in this species. This is because of the inter-species variability in the amino acid sequence of this protein and thus the low cross reactivity of commercially available antibodies. To aid in the development of such an assay, Vtg proteins in plasma were identified and characterized from 10 adult female alligators collected during the peak follicular period (from Lake Griffin, FL and Rockefeller State Wildlife Refuge, LA). Two sites were chosen in an ix

PAGE 10

effort to reduce site-specific bias. In addition, these sites were chosen for the ecological significance and environmental concerns associated with alligators in these geographical locations. Our study was designed to develop a qualitative method for identifying follicular females and for assessing their reproductive status; and to identify and characterize specific plasma proteins likely to be Vtg. Sodium dodecal sulfate-polyacrylimide gel electrophoresis (SDS-Page) analysis of plasma revealed three prominent protein bands unique to follicular females at ~250 to 450 kilo-daltons (similar to published molecular weights for Vtg or Vtg metabolites from other oviparous species). Further characterization of these proteins revealed that they were highly glycosylated and contained several phosphoserine amino acids. These proteins were isolated by SDS-Page and then confirmed by protein sequencing to have substantial homology with published Vtg sequences from other species. These data are critical for future development of an alligator specific Vtg assay. Such an assay could be used to further identify a possible mechanism for reproductive failures experienced by alligator populations in contaminant-impacted environments. Anatomical evaluations of reproductive status validated the plasma protein screening protocol. There was a 1:1 correlation for vitellogenic females exhibiting plasma proteins in the 250 to 450 kDa range. This correlation provided significant evidence that this is an acceptable method for discerning Vtg animals from non-Vtg animals. Each animal that had the three highly expressed plasma proteins also had a larger number of follicles in the 21 mm to 25 mm or > 26 mm size classifications. x

PAGE 11

CHAPTER 1 INTRODUCTION Heightened awareness of endocrine disruption (ED) in wildlife is expanding to a concern for humans as well. Many studies have helped to elucidate factors that may lead to the current state of environmental ED. These studies have focused on several environmental contaminants and their potential effects on many species of fish, birds, amphibians, and reptiles. While these studies have been useful in identifying several potential mechanisms of action for ED in these species, they have not completely fulfilled the purpose of representing the long-term impact on the health of the entire ecosystem. An animal model that could serve such a purpose needs to have a reasonable longevity to age-of-sexual-maturity ratio, and an upper-level predatory status in the food chain. Most bird, fish, amphibian, and reptilian species do not meet these criteria because they are sexually mature relatively soon after birth; and while they may be predators, they do not hold a very high place in the food chain. American alligators (Alligator mississippiensis) on the other hand, are upper-level predators, have an average life span of 50 years, and reach sexual maturity at 10 to 12 years. Their oviparous nature coupled with relatively long egg incubations make them an excellent model for studying reptilian embryonic development. Comprehensive embryonic studies have been conducted in normal alligator populations (including palatogenesis and hemoglobin amino-acid sequence development) (Densmore 1981, Le Clercq et al. 1981, Perutz et al. 1981, Ferguson 1985). For these and other reasons, the alligator is becoming a popular model 1

PAGE 12

2 for studying ED in the environment; and subsequently for making some preliminary predictions for potential concerns for human exposure. Because of all of these attributes, we chose the American alligator as our model for the development of biomarkers to assess ED. The following work describes the development of some of these techniques. Alligator Reproductive Anatomy and Physiology Much of the initial information obtained on the American alligator reproductive cycle in the southeastern United States comes from studies conducted on both wild and captive populations by various investigators. Their compiled findings are summarized, beginning with sexual maturity and ending with female reproductive anatomy. Our primary focus was on Florida and Louisiana studies in order to compliment this study. These data serve as a general overview of what is known about alligator reproduction. Age/Size of Sexual Maturity and Sex Ratios There is some discrepancy as to when a wild alligator reaches sexual maturity. The general consensus is that sexual maturity depends on size, which depends on environmental factors (e.g., temperature and food availability) that affect growth rates. However, other factors may influence sexual maturity (such as age, genetic differences between populations, and population densities) (Ferguson 1985). While there are published methods using bone annual growth zones for aging (Peabody 1961, Hutton 1986), they are not applicable to females because the bone remodeling females undergo during egg shell formation (Wink and Elsey 1986). Some general guidelines: Louisiana males and females both reach sexual maturity at 1.8 mo or 9 to 10 y (Joanen and McNease 1980); North Carolina males at 1.8 mo or 15 y, and females at 1.8 mo or 18 y (Ferguson 1985); and in southern Florida at 12 to 14 y (Dalrymple 1996). Some believe that age/size is not the only determining factor; that social order is also important. For

PAGE 13

3 example, larger, dominant males (longer than 2.7 m) are more likely to breed (Joanen and McNease 1980). These guidelines have been determined through survey analyses and may have intrinsic errors due to sampling and/or difficulties assessing age. Captive animals would seem to be an excellent control for these errors. However, animals reared in captivity develop variable growth patterns due to differences in environmental factors such as food quality and availability. This coupled with variations in mating behaviors, which develop from being in a constrained environment, leave these animals outside the norm and therefore unsuitable for calculating an average age/size for sexual maturity. Sex ratios in wild populations have received some attention. They are calculated from survey data which again can have intrinsic errors due to sampling error and/or animal movement during the breeding season. Ferguson and Joanen (1982, 1983) tried to approach this issue from the hatchling perspective in a 4-year study; and found approximately five females to every male. However, open-water surveys show very different ratios, depending on the month of sampling. These variations are probably due to adult females remaining in the marsh, which is their preferred habitat (Ferguson 1985). Reproductive Behavior Courtship and mating. Typical mating rituals involve bellowing, head slapping, and snout and head rubbing (Garrick and Lang 1977, Joanen and McNease 1989, Vliet 1989), culminating with copulation and subsequent nest building and egg deposition. Specific timing of the breeding season can vary slightly depending on the geographical location. However, the following data summarized by Ferguson (1985) serve as a suitable model (similar time frames were confirmed by Guillette et al. 1997 in their Florida study). These time points are based on air temperature (which was believed to be the driving force); not on the length of day (note temperatures in parentheses at each time

PAGE 14

4 point) for this particular study. However, there is no conclusive proof that temperature is the only trigger involved. Many investigators believe it is a combination of factors. In Louisiana, both females and males begin moving to deep open-water courtship areas in the early part of March (13C) (Ferguson 1985). Light bellowing by males (a guttural noise made to attract a mate) is heard throughout the month of April and elevates to mild bellowing by the end of April (21C) which lasts until the middle of May (24C) when intense bellowing begins. During this entire time, females develop ova in the ovaries; while male spermatogenesis begins in the middle of May and lasts approximately 4 weeks. Copulation begins around the third week of May and continues through the second week of June when spermatogenesis is at its peak. Females then move to shallower waters, and nest construction begins. Nest construction (see next section for a description) is complete and eggs are deposited by the end of June (27 to 28C). The female remains to tend and protect the nest, until the next spring, when hatchlings are ready to leave the nest. Males and non breeding females on the other hand, move to deep open water for the remainder of the season, returning to their winter habitat by the middle of October (22C). Nest construction. The female is solely responsible for building the nest. It consists of twigs, mud, and other debris that is indigenous to the area. For example, Florida nests consist mainly of saw grass, mud, and cotton tail grass. Typically an experienced female will build a mounded den-type nest that has a tunnel-like entrance. She will guard this entrance until she hears the hatchlings chirping (still inside the egg) approximately 65 days past laying (at which time she will uncover the nest and begin tending to the hatchlings). The average number of eggs per nest is 38 in Louisiana

PAGE 15

5 (Joanen and McNease 1989) and 42 to 45 in Florida (Woodward et al. 1993, Masson 1995, Guillette et al. 1997). However, there are some areas in Florida that have less eggs per nest such as Orange Lake = 33 eggs; Paynes Prairie = 34 eggs (Woodward et al. 1992) and Everglades National Park = 30 eggs (Kushlan and Jacobson 1990). Nests are usually located near the edge of a marshy area just above the water line. This can be a problem in times of draught that are followed by heavy rains because many nests can be flooded out; as was the case in Orange Lake, FL the year this study was conducted (unpublished data). Reproductive Endocrinology The male reproductive season begins in early spring after an increase in circulating testosterone (T) concentrations (which peak at approximately 90 ng/mL in April/May) (Lance 1983, 1984). This is concurrent with the production of mature sperm that are then stored in the seminiferous tubes. Females also have a T surge that occurs simultaneous to an increase in 17-estradiol (E2); however the peak is less than 1/10th that of mature males (Lance, 1983). This surge of a predominantly male hormone in females is not surprising, since T is the precursor for E2. There is some discrepancy between the Louisiana and the Florida studies as to the onset of the reproductive season in females. In Louisiana, Joanen and McNease (1980) and Lance (1989) found that the alligator reproductive season begins in early spring with an increase in circulating E2 concentrations, which peak in April at approximately 700 pg/mL. Guillette et al. (1997) determined that Florida females appear to have a bi-phasic cycle beginning in the fall. This fall phase of increased E2 concentrations return to summer concentrations (200 pg/mL) sometime between November and February,

PAGE 16

6 however, there was no sampling during this time frame. Subsequently there is a second increase in E2 concentrations beginning in February peaking at approximately 600 pg/mL in April/May (Guillette et al. 1997). This second rise in E2 causes the follicles to increase in diameter from 5 mm to 40 mm by late May to early June. It is unclear whether these differences in E2 cycling between the Louisiana and Florida studies is due to geographical variations or if it is just due to the Florida study including more time points (Guillette and Milnes 2001). Vitellogenesis is actively going on during this time of elevated E2 concentrations, and Guillette et al. (1997) discussed the possibility that the fall increase in E2 concentration served to produce an initial wave of large follicles that would in turn provide more circulating E2 which is needed for rapid oviductal growth. The spring E2 concentrations decrease rapidly following ovulation in June. Subsequent to the decline in plasma E2 concentrations, there is a rise in plasma progesterone (P) concentrations beginning in April and peaking at 5 ng/mL in June. This elevated concentration of plasma P continues circulating until oviposition and the beginning of luteolysis in June/July when the P concentrations decrease to 1 ng/mL (Guillette et al. 1997). Lance (1989) found that corpora lutea granulosa cells stained positively for 3-hydroxysteroid dehydrogenase-isomerase (3-HSD) which is the enzyme responsible for the synthesis of P. It is possible that plasma P produced in the corpora lutea aids in maintaining gravidity as it does in other species (Guillette and Milnes 2001). Female Reproductive Histology and Anatomy There is a right and left side to the reproductive tract; the right being the larger of the two. However both sides are simultaneously involved during each breeding season.

PAGE 17

7 Follicles are formed and nurtured in both the right and left ovaries and passed through the oviducts, the conduits (with various functional zones) which extend to the exterior of the body through a single vaginal opening. Folliculogenesis has been described by Uribe and Guillette (2000) as being a series of stages which are summarized in Table 1-1. Uribe and Guillette (2000) concluded that based on their histological findings, stages IVI compared to those of other reptiles, while stages VIIIX more closely resembled birds. Other features which were similar to birds include ovarian lacunae and smooth muscle bundles surrounding the follicles. However, there were some characteristics which were unlike birds or other reptiles such as: yolk morphology (animal and vegetal pole differences); yolk platelet structure; and theca morphology. While this staging system has proved invaluable in evaluating the progress of folliculogenesis, late-stage variations are difficult to interpret. This is mainly due to the awkwardness of sectioning a 40 mm follicle for histological evaluations; they are very large with very little support tissue (Guillette and Milnes 2001). The anatomy, and functionality of the oviduct in the American alligator is more similar to birds rather than to other reptilians. However, in contrast to birds which completely finish one egg before ovulating the next, alligators exhibit a more simultaneous ovulation and shelling of the entire clutch which is similar to reptilians (Guillette and Milnes 2001).

PAGE 18

8 Table 1-1: Stages of folliculogenesis. Stage Oocyte Diameter (mean SE) Characteristics Stage I: Previtellogenesis 42.8 6.6 m Nucleus contains chromatin in diplotene stage of meiotic prophase I. Thick chromosomes visible. One nucleolus. Squamous cells begin to surround oocyte. Stage II: Previtellogenesis 73.8 6.9 m Nucleus contains lampbrush chromosomes and one nucleolus. Stage III: Previtellogenesis 267.3 43.3 m Nucleus contains lampbrush chromosomes and multiple nucleoli. Squamous cells completely surround oocyte; monolayer is referred to as granulosa. Stage IV: Previtellogenesis 486.7 70.1 m Zona pellucida at periphery of oocyte. Granulosa cells are cuboidal containing a nucleus. Theca has developed, comprised of fibroblasts. Stage V: Previtellogenesis 1.2 0.9 mm Zona pellucida is considerably thicker, consisting of two layers; an inner striated layer and an outer hyaline band. Stage VI: Vitellogenesis 3.1 0.9 mm Peripheral granules and centralized vacuoles in ooplasm. Theca has sinuses. Stage VII: Vitellogenesis 4.5 1.6 mm Granules and vacuoles have increased greatly in numbers. Vacuoles are much larger (up to 25 m), some containing yolk platelets. Stage VIII: Vitellogenesis 6.8 3.4 mm Regional animal and vegetal poles clearly visable. Zona pellucida 18-20 m and have well defined radiata and hyaline layers. Theca contains blood vessels, collagen fibers, and flattened lacunae. Stage IX: Vitellogenesis 19.4 5.9 mm Ooplasm is filled with large (90 m) yolk platelets. Stage X: Vitellogenesis 38.8 2.4 mm Yolk platelets continue to grow (160 m). Theca thickens to 180-200 m), containing muscle cells as well. Source: Summarized from Uribe and Guillette (2000).

PAGE 19

9 The oviduct of the American alligator has been described in some detail (Palmer and Guillette 1992, Buhi et al. 1999). It has been divided into seven distinct regions each serving different purposes in preparing the mature follicle for deposition as an egg: The uppermost section, the anterior infundibulum functions to receive the mature follicle. The posterior infundibullum and the uterine tube are muscular with mucosal folds and believed to function in albumen secretion. The utero-tubal junction is a transparent non-muscular, non-glandular section which connects the uterine tube to the anterior uterus. The anterior and posterior uterus is the site of eggshell membrane formation and eggshell calcification, respectively. Finally, the posterior uterus connects to the vagina where the egg exits the body. Vitellogenin as a Reproductive Biomarker of Endocrine Disruption Vitellogenin (Vtg) has been classified as a hormonally controlled precursor protein to several of the yolk proteins found in oviparous eggs (Ryffel 1978). Once liver Vtg production is stimulated by circulating E2, it is post-translationally modified and circulated to the blood capillaries surrounding the follicular theca and transferred to the developing oocytes by diffusion from the follicular theca and subsequent pinocytosis by the oocytes (Wahli et al. 1981). Once in the oocytes, Vtg is proteolytically cleaved into lipovitellin and phosvitin; however the number of cleavage products is not known for most species (Ryffel 1978, Wahli et al. 1981). Characteristically, it is a highly glycosylated phospho-lipoprotein. The molecular weight ranges from ~150 to 600 kilo-daltons (Kd) depending on the species (Heppel et al. 1995, Brown et al. 1997, Allner et al. 1999, Brion et al. 2000). For example, in the African clawed-frog (Xenopus laevis), it occurs in the form of a dimer consisting of two 200 Kd polypeptides (Wahli et al. 1981), whereas in the Kemps Ridley sea turtle (Lepidochelys kempi) the predominant Vtg protein appears at 200 Kd (Heck et al. 1997). Similarly, the isoelectric point (pI), the pH

PAGE 20

10 at which the net charge of the protein is zero, ranges from ~ 6 to 7 depending on the species (Kawahara et al. 1983, James and Oliver 1997, Roubel et al. 1997). These characteristics were used collectively in the design of this study to optimize the chances of correctly identifying and characterizing Vtg in the American alligator. Since Vtg is a maternally derived protein that is utilized by the embryo as a nutritional source, it is possible that any deviation or disruption of the pathway may alter embryo development. The full extent to which the developing embryo uses Vtg is not clearly understood, but if it could act as a carrier protein for xenobiotic chemicals, then it would stand to reason that enzymes used by the embryo to metabolize those chemicals could be turned on and the activity up-regulated. Metabolism is a complex process in that enzymes are developed to control more than one event. For example cytochrome P450 enzymes are instrumental in xenobiotics metabolism as well as steroid metabolism (Ertl et al. 1999, Sierra-Santoyo et al. 2000). With this in mind it is possible that other events in the developing embryo could be affected by this exposure. There are several potential pathways and functions to explore but first there must be a definitive method for identifying and characterizing Vtg in the species being studied. This has been done for fish and birds, but there is limited information in amphibians other than Xenopus and reptiles. Vtg has been proposed as a biomarker of exposure to endocrine disrupting chemicals (EDC) in oviparous species (Sumpter and Jobling 1995). The rationale behind using Vtg as a biomarker stems from extensive research using the African clawed frog and the chicken (Gallus domesticus) as models for investigating E2 induced Vtg gene activation (Ryffel 1978). Studies on the Japenese medaka (Oryzias latipes) revealed that

PAGE 21

11 Vtg may be induced in males by E2 and EDC to produce Vtg at a level previously determined to be indicative of a reproductive female (Gronen et al. 1999). More recently, Vtg has been investigated in Florida as a biomarker of potential endocrine disrupting effects in largemouth bass (Micropterus salmoides) (Bowman et al. 2002, Seplveda et al. 2002). Numerous studies have been conducted in other species to identify and characterize this class of proteins (Wang and Williams 1982, Wahli et al. 1989, Hartling et al. 1997) and while there has been some work done in reptilian species such as lizards and turtles (Baerga-Santini and Morales 1991, Brown et al. 1997, Morales et al. 2002, Romano et al. 2002), there is very little reported for the crocodilians (Guillette et al. 1997). The reptilians which have been investigated most conclusively in this respect are turtles and lizards. The following is a brief summary of the most recent studies published. In the past 3 y there have been three comprehensive studies which have been essential in advancing turtle Vtg research to the point where quantitative assays are now possible. Duggan et al. (2001) analyzed plasma from the freshwater painted turtle (Chrysemys picta) in a seasonal study to fully characterize seasonal lipid transport in this species. They concluded that in this species, lipids and proteins control seasonal ovarian growth probably under hormonal control. These authors provide a detailed protocol for monitoring plasma lipids in turtles which utilized several techniques including the well known gravimetric method for total lipids as well as enzyme-linked immunosorbant assay (ELISA) methods for individual lipid components. Irwin et al. (2001) designed a study to analyze the potential effects of xenoestrogens present in cattle farm pond water on Vtg induction in the painted turtle. The rationale behind this study was that the

PAGE 22

12 manure runoff into the ponds could be carrying metabolized (glucoronide-conjugated) hormones which bacteria in the water could subsequently cleave into active steroids. These in turn could potentially induce the turtles and fish (male and female) that inhabited the ponds to increase hepatic Vtg production, therefore altering their reproductive cycles. They used an ELISA method designed to measure Vtg in both males and females from the affected ponds and compared them to a control site. They found that water concentrations of xenoestrogens in the water were sufficient to induce Vtg production in females but not in males. Herbst et al. (2003) recently published a comprehensive study designed to analyze the Vtg protein sequence in green turtles (Chelonia mydas) and compared it to published sequences from other species (tuatara [Sphenodon punctatus], chicken, and frog). They found that the n-terminal sequence obtained from 15 cycles of Edman degradation protein sequencing was not an exact match to anything in the National Center for Biotechnology Information (NCBI) or the Expressed Sequence Tags (EST) databases. The sequence however, had 73% homology with that of the tuatara (Brown et al. 1997). They then purified plasma Vtg to produce polyclonal and monoclonal antibodies to egg yolk granules which was reactive to green turtle Vtg in both ELISA and Western blot analyses. Lizard Vtg research has advanced from mere identification and MW determination (Carnevali et al. 1991, Baerga-Santini and Hernandez de Morales 1991) to time course analysis beginning with hepatic induction and ending with deposition in the developing follicle (Morales et al. 1996). Another interesting study conducted by Morales and Sanchez (1996) continued on their time course studies and investigated the effects of captivity on anole (Anolis pulchellus) Vtg production and subsequent follicular

PAGE 23

13 deposition. They found that long term captivity stress induced cessation of Vtg production and circulation could be alleviated by low level E2 hormone replacement therapy within 72 96 h. Talent et al. (2002) and Brasfield et al. (2002) both published studies advocating lizards as a potential reptilian model for ecotoxicological risk assessments. Talent et al. (2002) designed an egg injection study which revealed that 17-ethinylestradiol (an estrogenic chemical) caused male embryo feminization by impeding the development of secondary sex characteristics. Brasfield et al. (2002) designed a study to aid in the development of a protocol which could potentially be used as a quantitave tool for monitoring Vtg in western fence lizards (Sceloporus occidentalis). This study utilized a direct Vtg ELISA method and compared it to an indirect plasma alkaline-labile phosphate (ALP) method previously used in invertebrates (Kernaghan et al. 2002) and fish (Gagn et al. 1998, 2000, 2001) as an indirect measure of Vtg. They concluded that there was a high correlation between the two methods and that the ALP method could be a suitable measure of plasma Vtg in fence lizards. Rosanova et al. (2002) contributed invaluable data to the Vtg field by identifying the MW and location in two liver subcellular fractions of several Vtg precursor proteins in the oviparous lizard (Podarcis sicula). This study utilized Western blot analysis to identify two proteins (84 and 70 kDa) located in the rough endoplasmic reticulum (RER) and four proteins (180, 150, 60, and 50 kDa) located in the smooth microsomal fraction. Romano et al. (2002) conducted a time course study on the oviparous lizard (Podarcis sicula) which followed the fate of lipovitellins and phosvitins previously identified in egg yolks over a course of 44 days from ovoposition. There were two

PAGE 24

14 lipovitellins at 110 and 116 kDa that remained constant in the yolk throughout the 44 day incubation. The phosvitin profile underwent various changes throughout the 44 day incubation periods; on day one there were four proteins detected at 50, 45, 29, and 14 kDa; on day 10 post ovoposition, the 29 kDa phosvitin was missing but a new one was detected at 6.5 kDa; on day 18, only two phosvitins were detected at 14 and 6.5 kDa; and finally at day 44, only the 6.5 kDa phosvitin was detected. This suggested that there was a continuous degredation of the phosphorylated proteins in the egg yolk over the course of incubation. The interpretation of this degradation was that the embryo needed to be supplied with amino acids and smaller proteins during its embryonic growth. This study confirmed a need for assays that are capable of tracking these specific phosphorylated proteins or protein fractions throughout the time course which extends from egg production in the adult female liver through the developmental period of the embryo if we are to begin to elucidate the effects that contaminants (which may cause oxidative damage and subsequent dephosphorylation) may have on the reproductive success of these and other reptilian species. Heppel et al. (1995) attempted to develop a universal Vtg ELISA that would be reactive with plasma Vtg from several species including a snake and tuatara. The authors found that Vtg was only two to three times higher in vitellogenic females when compared to males (lizards and snakes), while the fish female reactivity was three to 10 times higher than the males depending on the species. Previous efforts to examine Vtg proteins in crocodilians have been qualitative or semi-quantitative. Matter et al. (1998) attempted to modify the method developed by Palmer and Palmer (1995) to quantify Vtg in hatchling alligators. Briefly, they

PAGE 25

15 performed Western blot analyses of hatchling plasma utilizing a rabbit anti-Vtg antibody which was raised against red-eared turtle Vtg. They were unable to detect an induction of plasma Vtg in hatchlings; however this was probably due to their young age coupled with continued lipovitellin and phosvitin contribution from their yolk sac. Brown et al. (1997) utilized an antibody raised against tuatara (Sphenodon punctatus) in a western blot analysis of adult female alligator plasma successfully recognizing a specific protein at ~220 kDa which they presumed to be Vtg. However this was not expanded upon, since the subject of their study was the tuatara. There has not been a quantitative assay published to date that is sensitive and specific for crocodilians. The current study was designed to characterize and isolate Vtg in the American alligator as a critical step toward the development of a quantitative assay for this species. Background and Significance The American alligator was placed on the United States endangered species list in 1967 (Groonbridge 1987). At that time, it was an acceptable practice to allow unlimited harvesting of animals for the sale of meat, skins, and trinkets such as teeth, claws, and skutes. It was even acceptable to harvest hatchlings and sell them as pets. Alligator populations appeared to be diminishing, therefore monitoring of the species was begun to determine the extent of the threat for extinction. Now after years of monitoring, experts agree the species has made an important recovery and is no longer in danger of extinction (Wood et al. 1985, Woodruff et al. 1989). However, the monitoring program that was established opened a new venue for environmental research and alligators became a popular model for contaminant studies in the southeastern United States due to their place in the food chain, their longevity, and therefore their potential for bioaccumulation of xenobiotics (Hall and Henry 1992, Crain and Guilette 1998). In fact, it has been

PAGE 26

16 proposed that many contaminants alligators are exposed to may be EDs (Gross et al. 1994, Guillette et al. 1994, Crain et al. 1997, Guillette and Gunderson 2001, Guillette et al. 2002). Alligator research in this area was originally conducted on eggs to determine a potential relationship between contaminants and their effects on reproductive success. However, to date no clear relationship has been established between the level of contaminants found in the eggs and embryo survival (Heinz et al. 1991). Therefore, an increasing number of researchers have begun looking at the adult female for a better understanding of mechanism(s) behind altered reproductive success. Due to the many factors that contribute to growth and maturity in this species such as temperature, population density, and food availability and quality (Hutton 1987), it is nearly impossible to determine if an adult female is reproductively active and will lay eggs in a particular year based on anatomical size alone. This coupled with permit limitations has led to the need for developing a non-invasive tool for evaluating the reproductive status of adult female alligators. The development of such a tool was the primary goal of this work. A secondary goal was to begin to isolate and characterize plasma Vtg from this species. This is of importance because it will aid future studies in elucidating a potential mode(s) of action of EDC. Study Objectives The objectives of the present study were to 1. Develop a qualitative method for identifying follicular females, and to 2. Identify and characterize female specific plasma proteins likely to be Vtg.

PAGE 27

CHAPTER 2 ASSESSMENT OF REPRODUCTIVE STATUS IN FEMALE AMERICAN ALLIGATORS Alligators are a popular model for reptilian contaminant studies due to their predatory place in the food chain, their longevity, and therefore their potential for bioaccumulation of contaminants (Hall and Henry 1992, Crain and Guilette 1998). It has been proposed that many of the contaminants that alligators are exposed to may be EDs (Gross et al. 1994, Guillette et al. 1994, Crain et al. 1997, Guillette and Gunderson 2001, Guillette et al. 2002). Contaminant research in this species was originally conducted on alligator eggs to determine a potential relationship between contaminants and their effects on reproductive success. So far, no clear relationship has been established between the level of contaminants found in the eggs and embryo survival (Heinz et al. 1991). Therefore, an increasing number of researchers have begun looking at the adult female for a better understanding of the mechanism(s) behind altered reproductive success. Due to the many factors that contribute to growth and maturity in this species, such as temperature, population density, and food availability and quality (Hutton 1987), it is nearly impossible to determine if an adult female is reproductively active and will lay eggs in a particular year based on anatomical size alone. This, coupled with permit limitations (as in Florida) has led to the need for developing a non-invasive tool for evaluating the reproductive status of adult female alligators. To date there is no such protocol published for alligators. This study was designed to develop a novel plasma 17

PAGE 28

18 protein assay which could be utilized for the prediction of reproductive status in adult female alligators. Vitellogenin (Vtg) protein is produced in the livers of reproductive female alligators, circulated through the blood, and subsequently deposited in the developing follicles. Along with being a reliable predictor of gravid females, it has also been proposed as a biomarker of exposure to EDC in oviparous species (Sumpter and Jobling 1995). However, to date, there has not been a quantitative assay published that is sensitive and specific for crocodilians. Heppel et al. (1995) attempted to develop a universal Vtg ELISA that would be reactive with plasma Vtg from several species including reptiles, but found that it was not sensitive enough (for reptiles) to be considered a reliable assay. Matter et al. (1998) attempted to modify the Western blot developed by Palmer and Palmer (1995) to be used as a quantitative Vtg assay in hatchling alligators. They were unable to detect an induction of plasma Vtg in the hatchlings, however this was probably due to their young age coupled with continued lipovitellin and phosvitin contribution from the yolk sac. Another factor to consider is the potential non-specific reactivity of the antibody with non-Vtg proteins. The current study was therefore designed with the intent that the data obtained herein could be used to further the efforts in developing such an assay that would be sensitive and specific for alligators. The primary objective of this study was to screen several free-ranging alligator females and develop a reproducible method for evaluating reproductive status. Animals were screened initially for the presence of highly expressed plasma proteins specific to adult females in the 250 to 350 kDa. This is the predicted MW range for Vtg in other

PAGE 29

19 oviparous species (Heppel et al. 1995, Brown et al. 1997, Allner et al. 1999, Brion et al. 2000). This is a relatively non-invasive procedure which should decrease the incidents of sacrificing animals that dont fit the studys criteria. A secondary objective of this study was to develop a standardized necropsy protocol which could be used to quantitatively assess the reproductive tract and thus be a tool for use in comparative studies. Materials and Methods Study Sites Two sites were chosen in an effort to reduce site specific bias from being introduced into the individual experiments. Each site was chosen for its significance to the ecological and environmental concerns surrounding alligators in their respective geographical locations (see following sections for relevance of chosen sites). Lake Griffin, Florida. Florida Lakes in the Ocklawaha River Basin have been the subject of environmental concern for the past few decades (Benton and Douglas 1994, Marburger et al. 2002). In the 1980s, Lake Apopkas alligator population declined noticeably suggesting a potential association with organochlorine pesticides (OCP) (Guilette et al. 1995). There were several point sources responsible for OCP contamination in Lake Apopka and subsequently the entire basin (Benton and Douglas 1994, Marburger et al. 2002). Lake Griffin, located downstream of Lake Apopka (Figure. 2-1), has moderate to elevated OCP concentrations in alligator egg yolks and decreased egg viability (Rauschenberger et al. 2003). Rockefeller State Wildlife Refuge, Louisiana. Rockefeller Wildlife refuge was donated to the State of Louisiana in 1920, and it is comprised of 76,042 acres (this is down from the original 80,000 acres due to erosion) which border the Gulf of Mexico (Figure. 2-2). The Deed of Donation mandated that the land be maintained as a wildlife

PAGE 30

20 refuge, and that there would be no public or commercial fishing or trapping. In 1983 there was an amendment to allow sport fishing and commercial trapping for the purpose of generating revenue for education and public health. This was amended again in 1987 ceasing the surplus revenue (Louisiana Department of Wildlife and Fisheries). Since then, it has been maintained as a refuge and it is staffed by a team of scientists, conservation officers, and of course a maintenance crew. The research conducted at Rockefeller has been instrumental in many of the advancements made in alligator ranching and physiology. There is limited access allowed to the public with regulations that are strictly enforced. This refuge has become popular as a reference site for many studies due to its low levels of soil contaminant concentrations (Elsey et al. 1999, Davis et al. 2001, Cobb et al. 2002) and the reduced level of stress to wildlife. Animals Adult female alligators (1.8 2.1 m) were captured by noose according to IACUC guidelines. Captures were coordinated such that animals from each site were at equivalent points in their reproductive seasons: Rockefeller animals were captured in mid April and Lake Griffin animals were captured in early May (these dates were chosen to target animals which would be in the late vitellogenic (V) stage of their reproductive cycle). These time points were confirmed to be similar when eggs were collected and staged later in the season: Rockefeller embryos were collected June 14th and staged at day 7 on June 29th, and Lake Griffin embryos were collected and staged at day 12 on July 1st. Animals were held in a moist cool enclosure until they were screened for the study criteria described below. Those meeting the criteria were held for sacrifice and those which did not meet the criteria were returned to their place of capture and released.

PAGE 31

21 Plasma Samples Blood (10 mL) was drawn from the occipital sinus into a heparinized syringe and transferred to heparinized tubes. The blood was set on ice until it could be centrifuged at 1000 rpm for 20 min in a Beckman J6-HC centrifuge to separate plasma from the cellular fraction. Once separated the plasma was snap-frozen in 1 mL aliquots and stored at -80C. Female specific protein determination Sodium dodecal sulfate polyacrylamide gel elctrophoresis analysis was performed according to the method described by Laemmeli (1970) to screen plasma for the presence of female specific proteins in the predicted MW range (~250 to 450 kDa) for Vtg. A predetermined criteria was set to categorize the plasma profiles in the 250 to 450 kDa range as being (1) highly vitellogenic if the female specific protein bands were at least two to three times more intense than that of a positive control female or (2) weak to non-vitellogenic if the female specific proteins were less intense than those of the control female or not present at all. These intensities were measured utilizing one individuals gross visual judgment due to the nature of the field set-up and lack of availability of a scanning densitometer. The positive control female used for these and subsequent experiments had been implanted with a 180 day time release pellet containing 20 mg of E2 in September 2001. Subsequently, plasma was drawn in December 2001 and preserved according to the protocol described previously (unpublished data, Gross et al. 2001). Protein extractions. All chemicals utilized in this section were purchased from Sigma-Aldrich Company Corp., St Louis. MO, USA. Plasma samples (100 L) were clarified by spinning at 10,000 rpm for 5 min in an Eppendorf microcentrifuge (to

PAGE 32

22 remove cellular components). A surfactant extraction buffer (containing a protease inhibitor cocktail made up of 4-(2-aminoethyl)benzenesulfonyl fluoride [AEBSF]; ethylenediaminetetraacetate [EDTA]; Bestatin, L-trans-3-Carboxyoxiran-2-carbonyl-L-leucylagmatine [E-64]; Leupeptin; and Aprotinin) was applied to plasma samples to liberate and denature proteins. This was prepared from a 10x extraction buffer which consisted of 500 mM Tris-HCl pH 8.0, 100 mM EDTA pH 8.0, 5% Triton-X 100, 2% sodium dodecylsulfate (SDS), 5% sodium deoxycholate (DOC), and 20 L/mL protease inhibitor cocktail. The 10x buffer was added to 90 L of clarified plasma to give a 1x final concentration. Samples were kept on ice during extraction to minimize degradation. Protein assay. Chemicals, pre-cast gels, protein standards, and equipment utilized in this and subsequent electrophoresis sections were purchased from Bio-Rad Laboratories, Hercules, CA, USA. Extracted protein samples were quantified according to Bradford (1976) using the Bio-Rad Protein Assay kit. A 1:100 dilution of each plasma sample was quantified by measurement against a bovine serum albumin (BSA, supplied in kit) standard curve ranging from 0 to 40 g/mL total protein. The micro protein assay was performed by pipetting 160 L of each sample dilution and standard into a 96 well plate in triplicate. Subsequently 40 L of G250 protein dye reagent (supplied in kit) was added to each well. Plates were incubated at room temperature for 5 min and subsequently read on a Dynex MRX Microtiter Plate Reader at 595 nm. Dynex Revelation software was used to develop a standard curve and extrapolate sample protein concentrations. Sample preparation. For each animal, 20 g total plasma protein, was prepared by adding sample reducing buffer containing 12.5% 0.5 M Tris-HCL pH 6.8; 25%

PAGE 33

23 glycerol; 2% SDS; 5% -mercaptoethanol; and 1.25% bromophenol at 2 3 times the sample volume and boiling for 1 min to denature the protein. Electrophoresis. The denatured protein samples were loaded onto 7.5% acrylamide denaturing gels for maximum high MW separation and reasonable band definition. A 1x running buffer (25 mM Tris base, 250 mM glycine, and 0.1% SDS) was used to perform electrophoresis on a Bio-Rad Mini-gel II apparatus powered by a Bio-Rad Power Pac 200. Molecular weights were confirmed by comparison to denatured MW standards which were run simultaneously with samples on each gel. Subsequent to electrophoresis, gels were fixed and stained with coomassie brilliant blue (CBB) protein staining solution composed of 40% methanol, 10% acetic acid, and 1% CBB overnight at room temperature with gentle agitation. The next day they were washed in several changes of de-stain (40% methanol and 10% acetic acid) to remove unbound stain and equilibrated in double deionized water (ddH2O). The gels were then dried between two pieces of cellophane sheets in a Bio-Rad Air Dryer for preservation and subsequent documentation on a Bio-Rad GS-800 densitometer. Data analysis. SDS-Page gels were analyzed and qualitatively graded for intensity of female specific bands in the 250 to 450 kDa MW range. These intensities were measured utilizing one individuals gross visual judgment due to the nature of the field set-up and lack of availability of a scanning densitometer. Only those animals that presented intensity in all female specific bands (when compared to a male plasma pool and the positive control female plasma [described previously]) were considered to be highly folliculogenic and subsequently chosen for sacrifice (see Figure. 2-5). Circulating Hormone Concentrations Plasma samples were analyzed by a standard radioimmunoassay (RIA) procedure

PAGE 34

24 to determine circulating E2 and T. This is a competitive binding assay set up to allow competition between the animals plasma hormone and a radiolabeled standard hormone for the binding site of a protein antibody. The following is a summary of the method from Giroux (1998). Sample extractions. For each hormone assay, plasma (50 l) was extracted twice with 4 mL of ethyl ether in duplicate (two tubes, each containing 50 l of plasma from each animal) for each hormone assay. Tubes were then vortexed for 1 min and then incubated in a methanol/dry ice bath for 3 to 4 min to precipitate (freeze) the aqueous fraction of the plasma. The ether/lipophilic plasma fraction was poured into a 100 mm glass tube and placed on a Labconco evaporator for 10 to 15 min. This procedure was repeated using the same tubes, thereby concentrating the two extractions together. Hormone assays. Standard curves and samples were prepared as follows. Total count tube (TC): 350 L phosphate buffered saline with 1% gelatin and 0.01% sodium azide (PBSGA) was added to 100 L radioactive label to determine the upper limit of the radioactivity in the assay; non-specific binding tube (NSB): 350 L phosphate buffer was added to 100 L radioactive label to measure its reaction with the antibody; zero binding tube (BO), 250 L phosphate buffer was combined with 100 L radioactive label and 100 L antibody to determine maximum binding of the unlabeled Ab-Ag complex; standard curve tubes, 200 L phosphate buffer was combined with 100 L radioactive label and 100 L antibody and 50 L known steroid in eight tubes of increasing concentrations from 0 pg/mL to 20,000 pg/mL; extracted sample pellets, 250 L phosphate buffer was combined with 100 L radioactive label and 100 L antibody specific for either E2 or T. All tubes were incubated for 24 hours at 4C. The next day

PAGE 35

25 250 L charcoal dextran was added to all tubes except the TC tubes and subsequently centrifuged for 10 min at 3000 rpm and 4C in a Beckman J6-HC centrifuge to remove the unbound antibody. For each tube, 0.4 mL of the supernatant was taken off and added to 4 mL of Scintiverse scintillation fluid (Fisher Scientific, Fairlawn, NJ, USA) in scintillation vials (United Laboratory Plastics, St. Louis, MO, USA). Samples were counted on a Packard Tri-Carb scintillation counter (model 1600CA). Unknown samples were quantified against the standard curve using the Beckman EIA/RIA Immunofit. Necropsies Twenty adult female alligators were screened at each site. Of the 20 animals from each site, 10 highly V and 3-5 weak to non-vitellogenic (NV) animals (for contrast) were sacrificed and necropsied. Anatomical reproductive tract evaluations were performed according to a standardized protocol (Table 2-1 and 2-2). Linear measurements (total length: tip of nose to tip of tail; snout-vent length; head length; and tail girth which was measured just behind the vent) were performed using a centimeter tape. Weight was determined by suspending the animal from a kilogram scale which was attached to a fork lift. Animals were sacrificed by cervical dislocation and double pithing. Subsequently, necropsies were performed according to the following protocol saving appropriate tissues for further analysis. The abdominal cavity was exposed by making two transverse cuts: one at the vent and one just below the chest cavity. Subsequently a longitudinal cut was made on one side at the transition between the dorsal and ventral side. The outer skin and fat layer was then filleted away from the abdominal membrane and the flap retracted. The abdominal cavity was further exposed by cutting away the rib cage and through the tough outer membrane. Once inside the cavity, organs were dissected out, weighed, and

PAGE 36

26 measured. The liver was weighed on a gram scale, and color and condition noted. The entire reproductive tract was removed from both the right and left sides. Photo-documentation was performed using a centimeter ruler for scale. The oviduct and ovaries were separated, weighed and measured (oviductal diameters were taken in the center of each anatomical section [defined in Chapter 1], lengths were not recorded due to expected inaccuracies subsequent to stretching and straightening). All follicles greater than 5 mm were counted, weighed, and measured using a gram scale and digital calipers. Health and reproductive parameters were evaluated using the following formulas: Condition factor; K = 100 x (weight (g)/length (cm)3) Hepatic Somatic index; HSI = 100 x (liver wt/body wt liver wt) Gonadal somatic index; GSI = 100 x (gonad wt/body wt gonad wt) Statistics were run for mean, SEM, equality of variance, and ANOVA (for multiple groups with sites) or T-tests (for individual means between sites) when appropriate using the Minitab statistical package. Results SDS-Page analyses revealed three bands in the Vtg MW range (~250, 350, and 450 kDa) that were present in higher concentrations in follicular animals (indicated by brackets in Figure. 2-3). These results correlated well (10 animals out of 10) with anatomical evaluations (Figure 2-4 panels A & B). Each animal from both sites that presented intense plasma protein bands in the above mentioned MW range (Fig 2-3 panel A) also presented a highly follicular (a greater number of large [>20 mm] follicles) reproductive tract (Fig 2-4 panel A & B). Conversely, each animal from both sites that presented weak to non-existent plasma protein bands in the above mentioned MW range (Fig 2-3 panel B) also presented a weakly follicular (a greater number of small [<20 mm]

PAGE 37

27 follicles) reproductive tract (Fig 2-4 panel C & D). Table 2-1 summarizes the average anatomical evaluations of all animals from both sites that were necropsied. Overall, Lake Griffin (LG) V and NV females were significantly larger (snout-vent length, head length, tail girth, and weight) when compared to Rockefeller (R) animals. Lake Griffin V females had a significantly higher condition factor when compared to R V animals but this was not true for the NV females when sites were compared. While there were significant differences noted between sites for the previously mentioned lengths (indicated in parenthesis), the total lengths were not significant. However, there was an overall trend for the LG animals to be longer than the R animals. The Rockefeller V animals had a significantly higher hepatosomatic index (HSI) when compared to LG V animals but this was not true for the NV females. There were no significant differences for any of the previously mentioned parameters noted when the V animals were compared to the NV animals within sites. Table 2-2 summarizes the average reproductive evaluations of all animals from both sites that were necropsied. Lake Griffin V animals had significantly larger oviductal weights and diameters when compared to R V animals. However, there were no significant differences noted for the oviductal measurements or ovarian weights for the NV females. The LG V females however had a significantly higher GSI compared to the R V animals while there was no significant difference between sites for the NV animals. The average numbers of follicles (overall totals and size class totals) were summarized in Table 2-2. There were no significant differences in the overall number of follicles when LG V animals are compared to R V animals. However, there were differences in the distribution of these follicles in the different size classes. For instance,

PAGE 38

28 LG V animals had significantly more 5 mm follicles in the right ovary and also contained follicles in the > 26 mm category, which was absent in the R V females, and R V animals had significantly more 16 mm follicles in both ovaries when compared to the LG animals. The distribution and frequency of the follicular size classes for each of the V animals are summarized for the two sites separately in Fig 2-5. These graphs reiterate the results obtained from the averages determined in Table 2-2. Overall, the LG V animals had a large number of predominantly > 26 mm follicles (Fig 2-5 panels C & D); while the R V animals had a larger number 16 mm and 21 mm follicles (Fig 2-5, panels A and B). In summary, when comparing reproductive tract measurements of V animals across sites, LG animals had significantly larger tracts containing a greater number of large follicles (> 26 mm). However, when comparing NV animals across sites (for all of the above reproductive measurements), there were only two significant differences noted: LG NV animals had significantly more 5 mm follicles, whereas R NV animals had significantly more 16 mm follicles. When the V animals were compared to the NV animals within sites (Table 2-2), the following significant differences were noted for the previously described reproductive measurements: LG V animals had significantly larger and heavier oviducts and ovaries than the LG NV animals, and for both sites V animals had higher GSI compared to NV animals. In addition, the R V animals had significantly more follicles overall. Conversely, there was no significant difference noted for the LG animals due to high variability in the NV animals, however, the trend indicated that there were more total follicles in the LG V animals. When the follicle size classes were compared, R V

PAGE 39

29 animals had significantly more 5 mm, 11 mm and 21 mm follicles than the R NV animals. The LG V animals had significantly more follicles 21 and > 26 mm follicles, while the LG NV animals did not have any follicles of these size classes. The average plasma E2 concentrations were 432 39 ng/mL and 571 73 ng/mL for R and LG V animals, respectively. The circulating T concentrations were 219 119 ng/mL and 279 66 ng/mL for R and LG V animals, respectively. There was no significant difference between these values for either hormone across sites. There was no hormone analysis performed on the NV animals. This was due to technical difficulties that arose after the V animals had been analyzed. As stated previously, there was a direct correlation (10 out 10 animals for each site) between the three female specific bands noted on the SDS-Page analysis of the plasma and the physical appearance of the reproductive tract (Figures 2-3 and 2-4). Table 2-1. Mean standard error of body measurements. Rockefeller Lake Griffin V NV V NV Total length (cm) Snout-vent length (cm) Head length (cm) Tail girth (cm) Weight (kg) Condition factor Hepatosomatic Index 232 7 120 4 37 1 56 2 38 4 0.3 0.01 1.3 0.01a 224 10 118 5 36 1 53 3 36 7 0.3 0.02 1.1 0.2 245 8 133 3a 41 1a 66 3a 59 6a 0.4 0.02 a 0.9 0.04 247 4 154 26 40 0.4 a 66 1 a 56 4 0.4 0.01 0.9 0.1 a Indicates a significant difference between sites (p < 0.05). (vitellogenic (V) sample size: n = 10, for each site; non-vitellogenic (NV) sample size: n = 3, for each site)

PAGE 40

Table 2-2. Mean standard error of the mean for reproductive measurements. Rockefeller Lake Griffin V NV V NV Oviduct diameter (mm) Upper Middle Lower Oviduct weight (g/kg body weight) Ovary weight (g/kg body weight) Gonadal-somatic index: Total number of follicles 5-10 mm 11-15 mm 16-20 mm 21-25 mm > 26 mm Plasma Estrogen (pg/mL) Plasma Testosterone (pg/mL) Left 13 17 25 4.8.5 b 4.4.5 0.9.1 b 29 b 7 b 3 b 10a 9 b 0 432 219 Right 12 15 25 5.0.5 b 4.9.5 31 b 7 b 4 b 10a 11 b 0 Left 8 14 24 1.7.5 3.0.8 0.4.1 6 1 0 3 1 1 N/A N/A Right 8 11 24 1.8.9 3.2.9 5 0 1 4a 0 0 Left 21a 19 38a b 6.9.7a b 9.2.2a b 1.8.2a b 34 4 2 2 6 b 20a b 571 279 Right 19a b 18 b 39a b 7.3.8a b 8.7.9a b 34 5 3 1.5 6 b 19a b Left 11 12 25 2.1.7 1.0.2 0.9.1 13 8 5 0 0 0 N/A N/A Right 10 9 28 2.2.7 1.1.1 20 15 a 5 0 0 0 30 a Indicates a significant difference between sites (p < 0.05). b Indicates a significant difference between V and NV within sites (p < 0.05. Missing samples from two animals, therefore no SEM. N/A: there was no hormone data available for NV animals. (vitellogenic (V) sample size: n=10, for each site; non-vitellogenic (NV) sample size: n=3, for each site) 30

PAGE 41

31 Lake Griffin Lake A p o p ka Figure 2-1. Map of the Oklawaha River Basin, Florida. Arrow indicates Lake Griifin.

PAGE 42

32 Figure 2-2. Map showing location of Rockefeller State Wildlife Refuge. Only part of the refuge is shown where the study took place (indicated by arrow).

PAGE 43

33 A MW R1 R2 R3 G1 G2 G3 250 150 B MW R4 R5 G4 G5 250 150 Figure 2-3. SDS-PAGE analysis of plasma samples from adult female alligator plasma. Brackets indicate expected molecular weight (MW) range for V proteins. lane contains plasma from an E2 induced control female. lane contains plasma from a control adult male alligator pool. Analysis normalized to total protein loaded. A) Vitellogenic (V) (n =3 from Lake Griffin [G] and Rockefeller [R]). B) Non-vitellogenic (NV) (n = 2 from Lake Griffin [G] and Rockefeller [R]). D C B A Figure 2-4. Photographic documentation of reproductive tracts of representative animals from each site. A) V Rockefeller female. B) V Lake Griffin female. C) NV pre-ovulatory Rockefeller female. D) NV pre-ovulatory Lake Griffin female.

PAGE 44

R1 to R10 Left Ovary01020304050Total5 1011 1516 2021 2526+ A G1 to G10 Left Ovary01020304050Total5 1011 1516 2021 2526+ C R1 to R10 Right Ovary01020304050Total5 1011 1516 2021 2526+ B G1 to G10 Right Ovary01020304050Total5 1011 1516 2021 2526+ D 34 Figure 2-5: Follicular frequency distribution in right and left ovaries. Each bar represents one female alligator. Y axis: The number of follicles in each size classification. X axis: Total (total number of follicles for each anim al), followed by each size classification (measured in mm). 34

PAGE 45

35 Discussion The three female specific proteins that were identified by this study have provided a good starting place for investigating plasma Vtg in alligators. They are within the predicted MW range based on information that has been published for other species, but it must be confirmed that one or all of these proteins are truly Vtg (see Chapter 3). Secondly, a full molecular characterization must be done to determine their origination and subsequent fate such as follicular deposition. The anatomical and reproductive evaluation section of this study was designed to serve three purposes: (1) to validate the results of the plasma protein screening; (2) to provide a comprehensive data base of anatomical and reproductive parameters of both Vtg and non-Vtg adult females; and (3) to use the data generated to develop a standardized protocol to be used for future evaluations of female alligators health and reproductive status. The evaluation of reproductive status validated the plasma protein screening protocol. There was a 1:1 correlation for V females exhibiting plasma proteins in the 250 to 450 kDa range. This correlation provided significant evidence that this is an acceptable method for discerning Vtg animals from non-Vtg animals. Each animal that had the three highly expressed plasma proteins also had a larger number of follicles in the 21 to 25 mm or > 26mm size classifications. There was also very little inter-animal variability in the oviductal diameters. This coupled with the low variability in their sizes (within sites) confers the likelihood that they were equivalent in age and gravida (number of times that they had reproduced). Alligator reproductive anatomy has been described comprehensively in the literature dating back as far as the 19th century. While there was no new information

PAGE 46

36 acquired from this study as far as reproductive anatomy, it did provide an essential teaching tool for this students education in reptilian anatomy. Subsequently, another opportunity arose in that a second investigator (Dr. Dave Rostal, Georgia Southern University) was simultaneously performing abdominal ultrasonography on the same animals used for this study in an effort to validate his method for use in alligators. There was a 1:1 correlation between Dr. Rostals results (predetermined criteria for a positive ultrasound was detection of follicles >15 mm) and the anatomical evaluations performed in this study. This collaboration proved to be beneficial to both groups while limiting the needless sacrifice of additional animals. The E2 hormone values were close to the expected average of 700 pg/mL for adult female alligators during the latter part of the reproductive cycle for both of these geographical locations. Testosterone values however, were well above the published average of 90 ng/mL. A possible explanation for the discrepancy in the average T values obtained is that the T hormone assay was lacking in sensitivity and/or specificity for alligators. Comparing this studys E2 analysis with others in the literature showed it to be useful in providing another parameter to evaluate the point the animals were at in their reproductive status, however it may have been improved by also including P in the hormone profile. This study demonstrated that a qualitative analysis of female specific plasma protein in alligators was a useful and predictive measure of folliculogenesis. There was an increase in the number of follicles in LG animals which did not coincide with a higher level of plasma E2 confirming that E2 had already peaked for the season and that these animals were in late vitellogenesis. This also suggests that there may be a different, non

PAGE 47

37 hormonally induced pathway at work in LG animals driving them to produce a greater number of larger follicles. Another possibility is that the hormone assay performed in this study lacked sensitivity and/or specificity for alligator plasma. However, since the values were comparable to those in the literature, this is not a very likely explanation. The anatomical evaluations demonstrated that overall LG animals were significantly larger than R animals with a higher condition factor indicating that the LG animals probably had more body fat as well. This could be due to seasonal variations between the two sites as females begin to mobilize fat deposits while they progress through their reproductive season. This could be an explanation for the larger follicles present in the LG animals. It seems to be a plausible possibility that the R animals were going to go through the same process later in the season thereby placing them slightly behind the LG animals in their reproductive status, however the egg staging data suggests the opposite. The R animals were captured and subsequently eggs were collected 2 weeks earlier than LG animals. The equivalency which is suggested in the materials and methods section is probably skewed because the R eggs had a longer period of time between when they were collected and when they were set in the incubator due to transportation. Two possible explanations for the reproductive differences between these two sites are: the animals were at equivalent stages when they were sacrificed with R animals having begun their season earlier; or, perhaps it is due to the fact that R females lay smaller eggs. Rockefeller animals had a higher HSI than the LG animals suggesting that they were in the process of increased hepatic protein production. Since Vtg is a hepatic protein, then perhaps they were just behind the LG animals in their Vtg production which would explain why they had smaller follicles overall. A

PAGE 48

38 comprehensive time course following the production of Vtg coupled with plasma hormones would need to be done at both of these sites to fully understand the variations between these two sites. Having a comparison of non-Vtg and Vtg animals within these sites provided another piece of information vital to the study of reproductive biology. This study reflected that 50% of the screened population at each site would go on to reproduce that year. This compares to other studies which have found that 63 to 68% of the adult female population reproduce in a given year. It is difficult to determine the accuracy of this studys data since all 20 animals from each site were not sacrificed. However, based on the three non-Vtg animals from each site that were sacrificed, it is likely that the non-Vtg animals would not have gone onto reproduce that year. It is a widely accepted supposition that crocodilian reproduction is temperature driven coupled with water level of the nesting areas. These two factors are inherently seasonal for each geographical location. This makes the theory of a second wave of reproductive females very unlikely.

PAGE 49

CHAPTER 3 IDENTIFICATION AND CHARACTERIZATION OF HIGH MOLECULAR WEIGHT FEMALE SPECIFIC PLASMA PROTEIN BANDS Vitellogenin (Vtg) has been classified as a hormonally controlled precursor protein to several of the yolk proteins found in oviparous eggs (Ryffel 1978). Once Vtg production is stimulated by circulating estradiol (E2) in the liver, it is post-translationally modified and circulated to the blood capillaries surrounding the follicular theca and transferred to the developing oocytes by diffusion from the follicular theca and subsequent pinocytosis by the oocytes (Wahli et al. 1981). Once in the oocytes, Vtg is proteolytically cleaved into lipovitellin and phosvitin, however the number of cleavage products is not definitively known and varies between species (Ryffel 1978, Wahli et al. 1981). Characteristically, it is a highly glycosylated phospho-lipoprotein. The precursor protein (circulated through the plasma) MW ranges from ~150 to 600 kilo-daltons (kDa) depending on the species (Heppel et al. 1995, Brown et al. 1997, Allner et al. 1999, Brion et al. 2000). For example, in the African clawed-frog (Xenopus laevis), it occurs in the form of a dimer consisting of two 200 kDa polypeptides (Wahli et al. 1981), whereas in the Kemps Ridley sea turtle (Lepidochelys kempi) the predominant Vtg protein appears at 200 kDa (Heck et al. 1997). Similarly, the isoelectric focusing point (pI) ranges from ~ 6 to 7 depending on the species (Kawahara et al. 1983, James and Oliver 1997, Roubel et al. 1997). These characteristics were used collectively in the design of this study to optimize the chances of correctly identifying and characterizing Vtg in the American alligator. 39

PAGE 50

40 The most extensive characterization of Vtg is in fish, birds (mainly chickens and quail), and amphibians (mainly the African clawed frog). Although little is known about this protein in reptiles, this research is rapidly growing and it is gaining popularity as a model for environmental endocrine disruption. Since Vtg is a maternally derived protein that is utilized by the embryo as a nutritional source, it is possible that any deviation or disruption of the pathway may alter embryo development. Subsequently it has been proposed as a biomarker of exposure to endocrine disrupting chemicals in oviparous species (Sumpter and Jobling 1995). The rationale behind using Vtg as a biomarker stems from extensive research using the African clawed frog and the chicken(Gallus domesticus) as models for investigating estrogen induced Vtg gene activation (Ryffel 1978). Studies on the Japenese medaka (Oryzias latipes) revealed that Vtg may be induced in males by E2 and endocrine disrupting chemicals to produce Vtg at a level previously determined to be indicative of a reproductive female (Gronen et al. 1999). More recently, Vtg has been investigated in Florida as a biomarker of potential endocrine disrupting effects in largemouth bass (Micropterus salmoides) (Bowman et al. 2002, Seplveda et al. 2002). Numerous studies have been done in other species to identify and characterize this class of proteins (Wang and Williams 1982, Wahli et al. 1989, Hartling et al. 1997); and while there has been some work done in reptilian species (described above) such as lizards and turtles (Baerga-Santini and Hernandez de Morales 1991, Brown et al. 1997, Morales et al. 2002, Romano et al. 2002), there is very little reported for the crocodilians (Guillette et al. 1997). There has not been a quantitative assay published to date that is sensitive and specific for crocodilians. The current study was designed to characterize and isolate Vtg

PAGE 51

41 in the American alligator as a critical step toward the development of a quantitative assay for this species. The previous chapter identified three female specific plasma proteins in the 250 to 500 kDa MW range which were present in higher concentrations in folliculargenic animals. Therefore the objectives of this study were to identify and characterize those high MW plasma proteins. We tested the hypothesis that Vtg was represented by one of these three bands. Materials and Methods Study Sites Two sites (Rockefeller State Wildlife Refuge, Louisiana and Lake Griffin, Florida), were chosen in an effort to reduce site specific bias from being introduced into the individual experiments. Each site was chosen for its significance to the ecological and environmental concerns surrounding alligators in their respective geographical locations (see previous chapter for a description of sites). Animals Adult female alligators (1.8.1 m) were captured, euthanized, and necropsied according to IACUC guidelines as described in Chapter 2. Plasma samples were obtained and preserved as described in Chapter 2. Female Specific Protein Determination The following methods were performed as described in Chapter 2 with the following modifications. Chemicals, pre-cast gels, protein standards, and equipment utilized in this and subsequent electrophoresis sections were purchased from Bio-Rad Laboratories, Hercules, CA, USA, or from Sigma-Aldrich Company Corp., St Louis. MO, USA except where indicated otherwise.

PAGE 52

42 Protein extractions. Plasma samples (100 L) were clarified by spinning at 10,000 rpm for 5 min in an Eppendorf microcentrifuge (to remove RBCs and WBCs). A non-ionic surfactant extraction buffer (without inhibitor cocktail to allow for enzyme digestions) was applied to plasma samples to liberate and denature proteins. This was prepared from a 10x extraction buffer composed of 500 mM Tris-HCl pH 8.0, 100 mM EDTA pH 8.0, 5% Triton-X 100 (SDS and DOC (due to their ionic nature) were not included in this buffer to allow for proper isoelectric focusing (IEF) described in the following section). The 10x buffer was added to 90 l of clarified plasma to give a 1x final concentration. Samples were kept on ice during extraction and alliquotted prior to snap freezing and subsequent storage at -80C to minimize degradation. Protein assay. Extracted protein samples were quantified according to Bradford (1976) using the Bio-Rad Protein Assay kit previously described in Chapter 2. Sample preparation. For each animal, 20 g total plasma protein, was prepared by the method previously described in Chapter 2. Electrophoresis. The denatured protein samples were then loaded onto 4 to 15% gradient acrylamide denaturing gels for maximum high MW separation while allowing for the capture of the entire protein profile from 250 kDa down to 25 kDa. Electrophoresis was then performed according to the method described previously in Chapter 2. Subsequently, gels were stained with coomassie brilliant blue for MW determination and dried between cellophane for documentation. A second set of gels were run simultaneously and stained for glycosylated proteins using a modified Periodic Acid-Schiff (PAS) method.

PAGE 53

43 Isoelectric focusing analysis. Semi-purified samples (prepared as follows) were utilized to determine the pI of the three female specific proteins to allow for a cleaner more focused analysis in lieu of conventional 2D-SDS Page analysis which can be very complex therefore limiting the ability to discern the protein of interest. The denatured protein samples (one animal chosen randomly from each site, extractions described previously) were loaded onto 7.5% acrylamide denaturing gels for maximum high MW separation (7 wells for each animal). Electrophoresis was then performed according to the method described in Chapter 2. The three female specific bands of interest were then excised and eluted in SDS-Page running buffer (7 slices for each band from each animal were combined in a separate elution tube) using the Bio-Rad model 422 Electro-eluter. This yielded 6 individual semi-purified samples; 1-250 kDa, 1-350 kDa, and 1-450 kDa protein for each of the Rockefeller and Lake Griffin animals. These samples were then concentrated using Centricon YM-100 spin columns (Millipore Corporation, Billerica, MA, USA) by centrifugation at 1000 rpm for 30 min. Subsequently, the samples were diluted to 2 mL in PBS and re-concentrated three times to exchange the buffer and remove the SDS. Samples (10 ng quantified by the protein assay described previously in Chapter 2) were combined with rehydration buffer (8 M urea, 2% 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate [CHAPS]; 40 mM dithiothreitol [DTT]; 0.2% Bio-LyteTM 3/10 ampholyte: Bio-Rad Ready/Prep 2-D starter kit), loaded onto pre-cast 7 cm immobilized pH gradient (IPG) strips (pH 5 to 8; Bio-Rad), and allowed to hydrate overnight. The strips were transferred to a clean, dry PROTEAN IEF focusing tray which was subsequently placed in the Bio-Rad PROTEAN IEF focusing cell and allowed to focus (covered with mineral oil) using the pre-set

PAGE 54

44 protocol (outlined below) determined by the length and number of strips. The strips were then stained with IEF stain from Bio-Rad (27% Isopropanol; 10% acetic acid; 0.04% coomassie blue R-250; 0.05% crocein scarlet) and subsequently destained with 50% methanol and 10% acetic acid until bands were discernable. The pI was noted and recorded. Pre-set protocol utilized to focus the proteins in the IEF focusing cell: Start voltage = 0 V End voltage = 8,000 V Volt-hours = 8-10,000 V-hr Ramp = rapid Temperature = 20C Enzyme Digests Plasma protein extractions described above were utilized to perform the following enzyme digests in an effort to confirm which (if not all) of the three female specific proteins contained phospho-lipid and sugar moieties. The digests were then analyzed by SDS-Page according to the method described above to determine any changes in MW of the three female specific proteins created by removing their covalently bonded groups. Deglycosylation. The E-DEGLY kit from Sigma was utilized to completely remove all N-linked and simple O-linked carbohydrates from the alligator plasma proteins. The kit contains the following enzymes; PNGase F (Chryseobacterium [Flavobacterium] meningosepticum) which cleaves all asparagine-linked complex, hybrid, or high mannose oligosaccharides (Tarentino et al. 1994) unless -core fucosylated (Szkudinski et al. 1995); -2(3,6,8,9) Neuraminidase (recombinant from Arthrobacter ureafaciens) which cleaves all non-reducing terminal branched and unbranched sialic acids (Uchida et al. 1979); O-Glycosidase (recombinant from

PAGE 55

45 Streptococcus pneumonia) which cleaves serine or threonine-linked unsubstituted Gal-(1-3)-GalNAc-(Glasgow et al. 1977, Iwase et al. 1993); (1-4)-Galactosidase (recombinant from Streptococcus pneumonia) which releases only (1-4)-linked, non-reducing terminal galactose (Glasgow et al. 1977); and -N-Acetylglucosaminidase (recombinant from Streptococcus pneumonia) which cleaves all non-reducing terminal -linked N-acetylglucosamine residues (Glasgow et al. 1977). For purposes of this study the following protocol was followed under denaturing conditions. Total plasma protein (100 g) was diluted to 30 l with deionized water (ddH20) in an Eppindorf tube, 10 l of 5x reaction buffer, 2.5 l of denaturation solution (both supplied in kit proprietary ingredients), 2.5 l of Triton X-100 solution, and 1 l of each enzyme (all in one tube to achieve complete deglycosylation) was added, mixed gently and incubated overnight at 37C. Subsequently 1/5th of this reaction was analyzed by SDS-Page on a 4-15% gradient gel, stained with CBB, dried, and scanned for photodocumentation (all described in chapter 2). Phospholipase digestion. Lipoprotein lipase (LPL) is found in vivo associated with heparin sulfate proteoglycans (HSPG) at the luminal surface of vascular endothelium (Olivecrona et al. 1993). It is essentially responsible for hydrolyzing triglycerides (TG) from very low density lipoprotein (VLDL) particles (Nilsson et al. 1980, Eckel 1989). Pruneta et al. (2001) isolated plasma VLDL and added exogenous bovine LPL to monitor the TG hydrolysis. The experiment described below was a modification of that study in that after the digestion, SDS-Page analysis was performed instead of monitoring the kinetics of the assay. Lipoprotein lipase (Sigma) was added (6 to 7 units/10 l) to 100 g total plasma protein (diluted to 10 l with ddH20) and 90 l

PAGE 56

46 1x reaction buffer (100 mM sodium phosphate, 150 mM sodium chloride, and 0.5% (v/v) Triton X-100; pH 7.2). Subsequently 1/5th of this reaction was analyzed by SDS-Page on a 4 15% gradient gel, stained with CBB, dried, and scanned for photo-documentation (all described in Chapter 2). Anti-Phospho-serine, -tyrosine, -threonine Western Blot Analysis Plasma protein extractions described above were utilized to perform the following Western blot analysis in an effort to confirm which (if not all) of the three female specific proteins were phosphorylated and to identify which of the three most likely phosphorylated amino acids they contained. Electrophoresis was performed as described previously. Subsequently the protein was transferred to a 0.45 m nitrocellulose membrane (Bio-Rad) for Western blot analysis. The protein transfer was accomplished by the following protocol optimized for the Bio-Rad mini trans-blot apparatus (Bio-Rad); gels, nitrocellulose membranes, whatman filter paper, and sponges were equilibrated for 15 minutes in transfer buffer (20% methanol in 25 mM Tris base, 250 mM glycine, and 0.01% SDS). Transfer sandwiches were then assembled in the following sequence; sponge on black side of holder, filter paper, gel, nitrocellulose, filter paper, and sponge. The holder was then locked and placed into the transfer module with the black side facing the black side of transfer module. The module was placed in the electrophoresis tank equipped with an ice block and filled with transfer buffer. Transfer proceeded at 90 volts constant for 2.5 hrs surrounded by ice to reduce chances of protein degradation due to overheating. Once the transfer was complete, the following immunoblot protocol was followed; membrane was rinsed quickly with ddH20 and subsequently blocked for 1 h in blocking buffer (5% BSA in PBS with 0.05% Tween-20 [PBS-T]); it was then incubated

PAGE 57

47 with constant agitation in a UVP HB-2000 Hybrilinker hybridation oven (Tango Technologies, Ltd., Boulder, CO, USA) in monoclonal primary antibody diluted in wash buffer (PBS-T) overnight at room temperature (RT) at the following dilutions: mouse anti-phosphoserine (Sigma) at 1:1000; mouse anti-phosphothreonine (Sigma) at 1:50; and mouse anti-phosphotyrosine (Sigma) at 1:2000. The next day the membrane was washed 3 x 5 minutes in wash buffer and subsequently incubated 1 hour at RT in goat anti-mouse IGG (alkaline phosphate conjugated) secondary antibody (Sigma) diluted to 1:30,000 in wash buffer. A final wash of 3 x 10 minutes with was buffer and 1x with ddH20 was performed prior to color development. Detection was performed by incubating the membrane in Western Blue Stabilized Substrate for Alkaline Phosphatase (Promega,) until bands of interest appear at desired intensity. This is a nitro blue tetazolium (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) substrate which turns a purple color when acted upon by alkaline phosphatase. Alkaline Labile Phospholipid (ALP) Analysis ALP analysis is an assay which has been used in some fish and invertebrate studies as an indirect assay for plasma or hemolypmph (respectively) Vtg. It was utilized in the current study as a secondary method to confirm the increased concentration of phospholipid proteins in the plasma of the highly vitellogenic females when compared to the weak to non-vitellogenic females. The method is described as follows: Plasma (350 L) was transfered to 16 x 125 mm glass tube containing 350 L tert-butyl methyl ether, mixed well, and incubated at room temp for 30 min (mixing every 10 min). This step extracted the lipophillic components (lipids and lipoproteins). The tubes were centrifuged in a J6-HC centrifuge for 2min at 3,000 rpm to ensure separation.

PAGE 58

48 The organic phase was transferred to a new tube and mixed with 100 L of 1 M NaOH and incubated at RT for 60 to 90 min (mixing well every 15 min). This step freed the alkali-labile phosphates. The tubes were centrifuged in a J6-HC centrifuge for 2 min at 3,000 rpm to ensure separation. The aqueous phase (containing the free phosphates) was transferred to a new tube for subsequent analysis by a modified (scaled down 1:5 to be performed in a 96 well format) phosphomolybdenum method (Phosphate Assay Kit; Sigma) which assays for inorganic phosphorous. Each sample (15 L in triplicate) was combined with development reagent consisting of TCA, molybdenum reagent, and Fiske-SubbaRow reducer and was quantified by measurement against an aqueous inorganic phosphorus standard curve (also in triplicate) ranging from 0 to 1.8 g/mL. A micro-protein assay was performed (as described previously) on the original plasma sample and results were utilized to report the ALP values in g of organic phosphate / mg protein. Amino-acid Sequencing Once the female specific proteins had been analyzed to determine that they had characteristics of Vtg from other species (correct MW, pI, phosphorylation, glycosylation, and phospholipid moieties), amino acid sequencing needed to be conducted to definitively identify them as Vtg. While there are publications that have utilized direct n-terminal sequencing by Edman degredation to identify Vtg, they have been marginally successful in obtaining full sequences. Therefore, for the purposes of this study internal polypeptide sequencing subsequent to enzyme digestion (of the whole protein) was chosen. The enzyme digestions reduced the protein to smaller polypeptide fragments thereby avoiding the covalently bonded groups that normally interfere with n-terminal sequencing. Plasma (25 g per well) from one animal from each site chosen

PAGE 59

49 randomly was electrophoresed as described previously in seven wells of a 7.5% polyacrylamide gel (a separate gel for each animal). The gels were stained with coomassie brilliant blue (as described previously), the three female specific bands in the 250 to 450 kDa range in each lane were excised along with a blank gel slice, same size bands combined as one sample (keeping bands from the two animals separate; yielding 6 samples three different size bands for each animal and 2 blank slices), and subsequently sent to the Interdisciplinary Center for Biotechnology Research (ICBR) Protein Sequencing Core for amino acid sequencing. The protocol performed by the Core is summarized below. In-gel digestion of proteins in polyacrylamide gel pieces. Each gel slice was cut into ~1 x 2 mm sections, placed into a 1.5 mL micro centrifuge tube with 150 L of 50% acetonitrile in 0.2 M ammonium bicarbonate (pH 8.9), and incubated for 30 min at 37C. This wash buffer was removed and the wash step repeated. The gel slices were then dried completely in a speed vacuum. Endoproteinase Asp-N enzyme (Roche Laboratyories,) solution was added to each sample using a 1:20 w/w ratio with 50 L 0.2 M ammonium bicarbonate (pH 8.9) and incubated for 24 h at 37C. The total volume of the sample (gel & buffer) was estimated and 45 mM DTT was added to give a final DTT concentration of 1 mM and subsequently incubated for 20 min at 50C. The samples were cooled to RT and an equal volume (to DTT volume) of 100 mM iodoacetic acid (IAA) was added and subsequently incubated for 20 min at RT in the dark. The supernatant was transferred to a new tube. The gel pieces were crushed and incubated for 30 min at RT with 100 L of 0.1% TFA / 60% acetonitrile. This extraction buffer was transferred to a filter tube and extraction was repeated combining the 2nd extraction with

PAGE 60

50 the first in the filter tube. Gel pieces were then discarded and the filter tube was centrifuged for 10 -15 min at maximum speed. The speed vacuum was used to decrease the final volume to < 150 L. This filtrate was then applied to an equilibrated Vydac C18 (= 2.1 x 150 mm, 300 pore size, and a 5 m particle size) reversed-phase HPLC column at 0.15 mL/min in 95% buffer A / 15% buffer B. Elution from the column was performed with buffer A / buffer B mixture according to the following gradient: 0-110 min (5% to 75% buffer B), and 110-120 min (75% to 85% buffer B). Elute was collected in 1.5 ml tubes which were capped immediately and stored at 4C until sequencing was performed. Protein sequencing. Repeated cycles of Edman degredation chemistry was utilized for n-terminal sequencing (on an Applied Biosystems model 494 HT Sequencer) of the polypeptides resulting from the enzyme digestions. Briefly, this entails the reaction of phenylisithiocyanate (PITC) with the n-terminal amino group of the polypeptide under mildly alkaline conditions to form an n-terminal PITC adduct. This was subsequently cleaved by anhydrous trifluoroacetic acid (TFA) yielding a thiazolinone derivative leaving the rest of the polypeptide intact. The thiazoline-amino acid was extracted into an organic solvent and subsequently treated with an aqueous acid to form a more stable phenylthiohydantoin (PTH) which was later identified by gas chromatography. Sixteen cycles were acquired with a sampling rate of 4.0 hz and detector scale of 1.0 AUFS. Results Three female specific bands were again detected by SDS-Page at ~250, 350, and 450 kDa (Figure 3-1). Upon isolation of the three bands (one Vtg plasma sample was

PAGE 61

51 chosen randomly from each site for this procedure), the pI was found to be ~7.2 for all three bands in both samples (data not shown). Glycosylation of the three female specific protein bands was determined by two methods: staining of an SDS-Page gel by a modified Periodic Acid-Schiff (PAS) method (Figure 3-2) and enzyme deglycosylation and subsequent analysis by SDS-Page to detect a shift in the electro-mobility (Figure 3-3). The PAS staining method was successful in identifying all three bands as being glycosylated in all 10 Vtg females from both sites (Figure 3-2 is a representative gel showing three animals from each site). This was further confirmed by enzyme deglycosylation of one Vtg plasma sample chosen randomly from each site and subsequent analysis SDS-Page (Figure 3-3). However the enzyme deglycosylation only showed an electrophoretic shift in the 250 kDa protein. An indirect method for the quantification of phospholipids was used initially to establish that there was a higher concentration of these lipophilic molecules in the plasma of Vtg females when compared to non-Vtg females. There was a significantly higher amount of phospholipid protein in the plasma samples of the Vtg females when compared to a male plasma pool, however there was no significant difference noted when sites were compared (Figure 3-4). These results were not strengthened by digesting one Vtg plasma sample from each site with phospholipase and subsequent analysis by SDS-Page (Figure 3-5). There was no significant electrophoretic shift noted in the 250 to 450 kDa proteins. This enzyme only digests phospholipid moieties, it will not digest a phosphorylated amino acid. Western blot analysis was used to identify which of the three bands (if not all) were phosphorylated and which of the three most commonly phosphphorylated amino

PAGE 62

52 acids did these proteins contain. The anti-phosphoserine blots revealed that the 250, 350 and the 450 kDa protein bands contained a high concentration of phosphorylated serines (Figure 3-6 Panel A). The anti-phosphotyrosine blots revealed that only the 250 kDa band contained phosphorylated tyrosine amino acids while the anti-phosphothreonine blots did not reveal any degree of phosphorylation in any of the three bands (Figure 3-6 Panel B and Panel C respectively). This was a third method confirming that the three proteins in the 250 to 450 kDa range are phosphorylated. Finally, while the sequencing project is still ongoing, preliminary results for the 250 kDa protein have revealed a 75 to 88 % homology when compared to published chicken, frog and fish Vtg sequences (see sequence alignments in Table 3-1). This is a small fragment resulting from the reconstruction of two out of five enzyme digest fractions of the 250 kDa protein from the Lake Griffin animal. There are 17 residues in this sequence with the highest confidence on residues 4-13. The sequence of this fragment is as follows; E (Glutamine) V (Valine) G (Glycine) I (Isoleucine) R (Argenine) A (Alanine) E (Glutamine) G (Glycine) L (Leucine) G (Glycine) X (unidentified). A sequence homology search was performed utilizing the Basic Local Alignment Search Tool (BLAST) which is provided through the National Center for Biotechnology Information (NCBI) server. Of the 100 sequences that were returned in the query, 11 of them were Vtg sequences from various species of chickens, frogs, and fish. Discussion This study was designed to meet the following objectives: (1) isolate and characterize the three female specific bands that had been identified in the previous screening study and (2) use what little is known in the literature about alligator Vtg to prove that one or all of those three bands are or are not Vtg.

PAGE 63

53 Characteristically Vtg has been proven to be a highly glycosylated phospholipid protein in other species. The published MW weight ranges from 150 to 600 kDa depending on the species being investigated. There have also been some lower MW products which have been referred to as Vtg-like. Vtg is a complex protein that originates in the liver of oviparous vertebrates. Much of the confusion in regards to the actual size of the protein is probably due to the fact that it undergoes extensive post-translational processing upon transfer out of the liver as well as after it begins its journey through the bloodstream and then again prior to being taken up by the oocytes. The form that shows up in an assay is dependant on many factors including the reproductive status of the animal being tested. Vtg production and modification can be affected by hormonal influences, diet, and other environmental factors including seasonal changes. Taking all of this into account, the present study was designed to target the most likely candidates and analyze them for characteristics specific to Vtg or Vtg-like proteins. Secondly, utilize amino acid sequencing and submit this data to the available sequence banks as a method to identify Vtg. The initial characterization of the three female specific proteins can be summarized as follows: (1) The 250 kDa protein is highly glycosylated and contains several phosphorylated serine amino acids as well as some phosphorylated tyrosines. The phospholipase digestion showed no electrophoretic mobility shift indicating that there was very little (or no) phospholipid present. The pI is ~7.2 which is in the predicted range for Vtg. (2) The 350 kDa protein is highly glycosylated and contains several phosphorylated serine amino acids as well as some phosphorylated tyrosines. The phospholipase digestion did not show an electrophoretic mobility shift indicating that

PAGE 64

54 there were very little (or no) phospholipid moieties present. The pI is ~7.2 which is in the predicted range for Vtg. (3) The 450 kDa protein is glycosylated but to a lesser degree than the other two proteins. It contains several phosphorylated serine amino acids. The phospholipase digestion showed minimal electrophoretic mobility shift indicating that there was very little (or no) phospholipid present. The pI is ~7.2 which is in the predicted range for Vtg. Another explanation for the failed phospholipase digestion could be that the reaction is just not sensitive enough to discern the phospholipid moieties due to the complex nature of the Vtg protein. It is possible that these moieties are protected in the folding of the protein which would not allow for proper digestion by the phospholipase enzyme. The preliminary amino acid sequencing revealed that the nine residues obtained from the 250 kDa protein have 75 to 88 % homology with published sequences from chickens, frogs, and fish. This data coupled with the characterization described previously infers that the 250 kDa female specific protein identified in this study is probably Vtg. Conclusion of the sequencing project should provide sufficient evidence to confirm this and to identify the other two female specific proteins as well.

PAGE 65

Table 3-1: Amino acid sequence alignment resulting from BLAST search. Query represents the nine residues obtained from the 250 kDa female specific protein isolated from American alligator (Alligator mississippiensis) plasma. Species Start Sequence End % homology to query sequence Reference QUERY 1 E V G I R A E G L 9 Chicken (Gallus gallus ) 679 E V G I R V E G L 687 88 % Walker et al., 1983 Chicken (Gallus gallus ) 679 E V G I A A E G L 687 88 % Yamamura et al., 1995 African clawed frog (Xenopus laevis ) 681 E I G I R G E G 688 75 % Walker et al., 1984 African clawed frog (Xenopus laevis ) 681 E V A L R A E G L 689 77 % Yoshitome, 2003 Japanese whiting (Sillago japonica ) 679 E V G V R A E G 686 87 % Yoon, 2002 Blue tilapia (Oreochromis aureus ) 679 E V G V R T E G 686 75 % Lim et al., 1997 Rainbow Trout (Oncorhynchus mykiss ) 679 E V G V R T E G 686 75 % Le Guellec et al., 1988 Rainbow trout (Oncorhynchus mykiss ) 679 E V G V R T E G 686 75 % Mouchel et al., 1996 Zebrafish (Danio rerio ) 678 G I R A E G L 684 85 % Wang et al., 2000 Japenese medaka (Oryzias latipes) 680 E V G V R T E G 687 75 % Murakami & Nakai, 2001 CONCENSUS E V G R E G L 55 (-) Denotes missing amino acid. (*) Denotes lack of concensus. 55

PAGE 66

56 MW R1 R2 R3 G1 G2 G3 250 150 100 75 50 37 25 Figure 3-1. SDS-PAGE analysis of plasma samples from three Vtg adult female alligators. Three were from Lake Griffin [G] and three from Rockefeller [R]). Gel was stained with coomassie brilliant blue. Brackets surround expected molecular weight (MW) range for Vtg proteins. lane contains plasma from an E2 induced control female. lane contains plasma from a control male pool. Analysis normalized to total protein loaded. As was noted in Chapter 2 (Figure 2-3 panel A), there are three prominent bands in the 250 kDa MW range for both sites.

PAGE 67

57 1 00 1 50 2 50 MW R1 R2 R3 G1 G2 G3 7 5 50 3 7 Figure 3-2. Glycosylation analysis of plasma samples. SDS-PAGE analysis of plasma samples from three Vtg adult female alligators (three from Lake Griffin [G] and Rockefeller [R]) stained for glycosylation using a modified Periodic Acid-Schiff (PAS) method. Brackets surround expected molecular weight (MW) range for Vtg proteins. lane contains plasma from an E2 induced control female. lane contains plasma from a control male pool. Analysis normalized to total protein loaded. PAS stain only stains proteins that are glycosylated. It is clear that the three bands in the 250 kDa range are highly glycosylated in animals from both sites.

PAGE 68

58 F Fx BSA BSAx x R Rx G G1x MW BSA 250 150 100 75 50 37 Figure 3-3. Deglycosylation analysis of alligator plasma. SDS-PAGE analysis of plasma samples from two Vtg adult female alligators (one from each site) stained with coomassie brilliant blue. One of each sample was deglycosylated by enzyme digestion prior to being electrophoresed. Samples without enzyme are indicated by X. F lanes contain Feutin; protein positive for glycosylation. BSA was included as a negative control for glycosylation. Brackets surrounds expected molecular weight (MW) range for Vtg proteins. Successful deglycosylation is identified by the electrophoretic mobility of the protein shifting down indicating a lower MW. There was only slight deglycosylation noted in the 250 kDa protein for both the Lake Griffin and the Rockefeller animals.

PAGE 69

59 in ote pr g /m 4 ugPO 323 is male plasma value2505007501000Plasma Rock Griffin Figure 3-4. ALP analysis of plasma proteins. ALP analysis confirming the increased concentration of phospholipid proteins in the plasma of the vitellogenic females when compared to the male pool (indicated by horizontal line).

PAGE 70

60 MW BSA BSAx x R1 R1x G1 G1x 250 150 100 75 50 37 25 14 250 150 Figure 3-5. Phospholipase digestion of alligator plasma. SDS-PAGE analysis of plasma samples from two Vtg adult female alligators. One from Lake Griffin [G] and one from Rockefeller[R]) stained with coomassie brilliant blue. One of each sample was treated with phospholipase prior to being electrophoresed. Samples without enzyme are indicated by X. lane contains plasma from a control male pool. BSA was included as a negative control. Brackets surround expected molecular weight (MW) range for Vtg proteins. Successful dephosphorylation would be identified by the electrophoretic mobility of the protein shifting down indicating a lower MW. However, there was no dephosphorylation noted for either the Lake Griffin or the Rockefeller animal.

PAGE 71

61 250 150 100 75 50 MW BSA R1 G1 BSA R1 G1 BSA R1 G1 BSA R1 G1 37 A B C D Figure 3-6. Western blot analysis of phosphorylated proteins in alligator plasma. Western blot analysis analysis of plasma samples from two Vtg adult female alligators (one from Lake Griffin [G] and one from Rockefeller [R]). BSA was included as a negative control. Brackets surround expected molecular weight (MW) range for Vtg proteins. (A) Blot was incubated in -phospho-serine primary antibody. (B) Blot was incubated in -phospho-tyrosine primary antibody. (C) Blot was incubated in -phospho-threonine primary antibody. (D) Blot was incubated without primary antibody. -phospho-serine primary antibody reacted the strongest with all three proteins in the 250 kDa MW range while the -phospho-tyrosine primary antibody only reacted with the 250 kDa protein and the -phospho-threonine primary antibody did not react with any of the three proteins.

PAGE 72

CHAPTER 4 CONCLUSIONS AND FUTURE DIRECTIONS This is the first study that has attempted to identify Vtg in adult female American alligators through the utilization of sequence analysis coupled with limited biochemical characterization. It is therefore essential that additional sequencing be completed. These data will then be useful in developing a sensitive and specific quantitative assay for alligator Vtg. Such an assay could then be utilized throughout the entire reproductive cycle for several sites to establish a seasonal monitoring protocol. This could be expanded to a time course study designed to follow the production of Vtg and its subsequent modifications and eventual deposition in the growing follicles. There are several possible explanations which would support that the three female specific proteins analyzed in this study are or are not Vtg or Vtg metabolites. Based on the information gathered the most likely explanation is that they are Vtg metabolites (sequencing data confirms this to be true for the 250 kDa protein) that are at different stages of post-translational modification. It is likely that their inevitable fate will be deposition in the growing oocyte to be used by the embryo as a nutritional source. However, another possibility is that one or more of them are polypeptides which have been cleaved into one or more products upon analysis by denaturing SDS-Page. The phospholipase digestion analysis provided data that is in direct conflict with what has been previously described in the literature. As already discussed in Chapter 3, there are plausible explanations as to why this assay may have yielded negative results; complexity of the sample or lack of sensitivity of the assay. However there is another 62

PAGE 73

63 possibility; perhaps Vtg is constructed differently than we have all assumed. It is published many times over that Vtg is a phospholipoprotein. This implies that there are phosphorylated lipid moieties attached to the protein backbone. If this were true, and the negative results were not due to interference, then the phospholipase would have cleaved these moieties from the backbone leaving a smaller phospholipid product and the remainder of the protein construct as a second product. Perhaps Vtg is more complex than was previously assumed. The western blot analysis identifying phosphorylated amino acids confirms that there are phosphate groups attached directly to the protein backbone. Further investigation of the structure and subsequent folding of the protein is warranted. This type of research would help to elucidate potential structure activity relationships between Vtg and other proteins as well as EDCs such as OCPs. During the initial analysis of plasma protein profiles there were some subtle differences in lower MW proteins which did not fall within the target MW range for this study thereby suggesting qualitative differences in the post-translational processing of other female specific proteins in animals from Lake Griffin compared to Rockefeller. These results warrant further investigations of these plasma protein profiles from female animals from these sites as well as others to determine whether the differences may be contaminant related or whether they are just an artifact of regional genetic variations. Once this question is answered, it would be beneficial to examine the livers from the same animals looking specifically at Vtg precursors and other reproductive proteins (including metabolic enzymes) to begin to elucidate a potential mechanism(s) for metabolic alterations which may affect reproductive success in animals from OPC contaminated sites. Therefore future directions should include the same types of analysis

PAGE 74

64 on the liver and ovidutcal tissues to further enhance knowledge of the alligator reproductive system at the molecular level. To date this is an underdeveloped area which could help to elucidate the mechanism(s) behind altered reproductive success in these animals. Eventually there needs to be a binding assay developed in alligators which would be able to explore the interactions of Vtg and various tissues and subsequent involvement with other proteins such as potential carrier or chaperone proteins. This could be expanded to explore possible interaction of these proteins with OCPs and other EDCs.

PAGE 75

REFERENCE LIST Allner B, Wegener G, Knacker T, Stahlschmidt-Allner P (1999). Electrophoretic determination of estrogen-induced protein in fish exposed to synthetic and naturally occuring chemicals. Sci Total Environ. 233, 21-31. Baerga-Santini C and Hernandez de Morales M (1991). Vitellogenin diversity in tropical lizards (Anolis pulchellus): identification and partial characterization. Comp Biochem Physiol B Biochem Mol Biol. 100, 347-359. Benton J, Douglas D. (1994) Ocklawaha fisheries investigations completion report, 1991-1994: Study XIII, Assessment of fisheries restoration potential for reclaimed agricultural lands in the upper Ocklawaha Basin; Study XIV, Ocklawaha chain of lakes largemouth bass population studies; Study XV, Black crappie production in Lake Griffin. State of Florida Game and Fresh Water Fish Commission. Bowman CJ, Kroll KJ, Gross TG, Denslow ND (2002). Estradiol-induced gene expression in largemouth bass (Micropterus salmoides). Mol Cell Endocrinol. 196, 67-77. Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 7, 248-254. Bradsfield SM, Weber LP, Talent LG, Janz DM (2002). Dose-response and time course relationships for vitellogenin induction in male western fence lizards (Sceloporus occidentalis) exposed to ethinylestradiol. Environ Toxicol Chem. 21, 1410-1416. Brion F, Rogerieux F, Noury P, Migeon B, Flammarion P, Thybaud E, Porcher JM, (2000). Two-step purification method of vitellogenin from three teleost fish species: rainbow trout (Oncorhynchus mykiss), Gudgeon (Gobio gobio) and chub (Leuciscus cephalus). J Chromotogr B Biomed Sci Appl. 737, 3-12. Brown MA, Carne A, Chambers GK (1997). Purification partial characterization and peptide sequences of vitellogenin from a reptile, the tuatura (Sphenodon punctatus). Comp Biochem Physiol B Biochem Mol Biol. 117, 159-168. Buhi WC, Alvarez IM, Binelli M, Walworth ES, Guillette LJ Jr. (1999). Identification and characterization of proteins synthesized de novo and secreted by the reproductive tract of the American alligator, Alligator mississippiensis. J Reprod Fertil. 115, 201-213. 65

PAGE 76

66 Carnevali O, Mosconi G, Angelini F, Limatola E, Ciarcia G, Polzonetti-Magni A (1991). Plasma vitellogenin and 17 beta-estradiol levels during the annual reproductive cycle of Podacis s. sicula Raf. Gen and Comp Endocrinol. 84, 337-343. Cobb GP, Houlis PD, Bargar TA (2002). Polychlorinated biphenyl occurence in American alligators (Alligator mississippiensis) from Louisiana and South Carolina. Environ Pollut. 118, 1-4. Crain DA and Guillette LJ Jr. (1998). Reptiles as models of contaminant-induced endocrine disruption. Anim Reprod Sci. 53, 77-86. Crain DA, Guillette LJ Jr. Rooney AA Pickford DB (1997). Alterations in steroidogenesis in alligators (Alligator mississippiensis) exposed naturally and experimentally to environmental contaminants. Environ Health Perspect. 105, 528-533. Dalrymple GH (1996). Growth of American alligators in the Shark Valley region of Everglades National Park. Copeia. 212-216. Davis LM, Glenn TC, Elsey RM, Dessauers HC, Sawyer RH (2001). Multiple paternity and mating patterns in the American alligator, Alligator mississippiensis. Mol Ecol. 10, 1011-1024. Densmore LD. (1981). Biochemical and immunologicalsystematics of the order crocodilia. New Orleans, Louisiana State University. Duggan A, Paolucci M, Tercyak A, Gigliotti M, Small D, Callard I (2001). Seasonal variations in plasma lipids, lipoproteins, apolipoprotein A-I and Vitellogenin in the freshwater turtle, Chrysemys picta. Comp Biochem Physiol A Mol Integr Physiol. 130, 253-269. Eckel RH (1989). Lipoprotein lipase. A multifunctional enzyme relevant to common mtabolic diseases. N Engl J Med. 320, 1060-1068. Elsey RM, Lance VA, Campbell L (1999). Mercury levels in alligator meat in South Louisiana. Bull Environ Contam Toxicol. 63, 598-603. Ertl RP, Bandiera SM, Buhler DR, Stegman JJ, Winston GW (1999). Immunochemical analysis of liver microsomal cytochromes P450 of the American alligator, Alligator mississippiensis. Toxicol Appl Pharmacol. 157, 157-165. Ferguson MWJ (1985). Reproductive Biology and Embryology of the Crocodilians. In 'Biology of the Reptilia'. pp. 329-491. (John Wiley & Sons: New York.) Ferguson MWJ and Joanen T (1982). Temperature of egg incubation determines sex in Alligator mississippiensis. Nature London 296, 850-853. Ferguson MWJ and Joanen T (1983). Temperature dependent sex determination in Alligator mississippiensis. J Zool. 200, 143-177.

PAGE 77

67 Gagn F and Blaise C (1998). Estrogenic properties of municipal and industrial wastewaters evaluated with a rapid and sensitive chemoluminescent in situ hybridization assay (CISH) in rainbow trout hepatocytes. Aquat Toxicol. 44, 83-92. Gagn F and Blaise C (2000). Evaluation of environmental estrogens with a fish cell line. Bull Environ Contam Toxicol. 65, 494-500. Gagn F, Marcogliese DJ, Blaise C, Gendron AD (2001). Occurrence of compounds estrogenic to freshwater mussels in surface waters in an urban area. Environ Toxicol. 16, 260-268. Garrick LD and Lang JW (1977). Social signals and behaviors of adult alligators and crocodiles. Am Zool. 17, 225-239. Giroux DJ. (1998) Lake Apopka revisited: a correlational analysis of nesting anomalies and DDT contaminants. Gainesville, Florida, University of Florida. Glasgow LR, Paulson JC, Hill RL (1977). Systematic purification of five glycosidases from Streptococcus (Diplococcus) pneumoniae. J Biol Chem. 252, 8615-8623. Gronen S, Denslow N, Manning S, Barnes S, Barnes D, Brouwer M (1999). Serum vitellogenin levels and reproductive impairment of male Japanes medaka (Oryzias latipes) exposed to 4-tert-octylphenol. Environ Health Perspect. 107, 385-390. Groombridge B (1987). The distribution and status of world crocodilians. In 'Wildlife Management: Crocodiles and Alligators'. pp. 9-21. (Surrey Beatty and Sons: Australia.) Gross TS, Guillette LJ Jr., Percival HF, Masson GR, Matter JM, Woodward AR (1994). Contaminant-induced reproductive anomalies in Florida. Comp Pathol Bull. 26, 2-8. Guillette LJ Jr., Gross TS, Gross DA, Rooney AA, Percival HF (1995). Gonadal steroidogenesis in vitro from juvenile alligators obtained from contaminated or control lakes. Environ Health Perspect, Suppl. 103, 31-36. Guillette LJ Jr., Gross TS, Masson GR, Matter JM, Percival HF, Woodward AR (1994). Developmental abnormalities of the gonad and abnormal sex hormone concentrations in juvenile alligators from contaminated and control lakes in Florida. Environ Health Perspect. 102, 680-688. Guillette LJ Jr. and Gunderson MP (2001). Alterations in development of reproductive and endocrine systems of wildlife populations exposed to endocrine-disrupting contaminants. Reproduction 122, 857-864. Guillette LJ Jr., Vonier PM, McLachlan JA (2002). Affinity of the alligator estrogen receptor for serum pesticide contaminants. Toxicology 181-182, 151-154.

PAGE 78

68 Guillette LJ Jr., Woodward AR, Crain DA, Masson GR, Palmer BD, Cox C, You-Xiang Q (1997). The reproductive cycle of the American alligator (Alligator mississippiensis). Gen Comp Endocrinol. 108, 87-101. Guillette LJ Jr. and Milnes MR (2001). Recent observations on the reproductive physiology and toxicology of crocodilians. In 'Crocodilian Biology and Evolution'. pp. 199-213. (Surrey Beatty and Sons: Australia.) Hall RJ and Henry PFP (1992). Assassing effects of pesticides on amphibians and reptiles: status and needs. Herpetol J. UK, 65-71. Hartling RC, Pereira JJ, Kunkel JG (1997). Characterization of a heat-stable fraction of lipovitellin and development of an immunoassay for vitellogenin and yolk protein in winter flounder (Pleuronectes americanus). J Exp Zool. 278, 156-166. Heck J, MacKenzie DS, Rostal D, Medler K (1997). Estrogen induction of plasma vitellogenin in the Kemp's Ridley sea turtle (Lepidochelys kempi). Gen Comp Endocrinol. 107, 280-288. Heinz GH, Percival HF, Jennings ML (1991). Contaminants in American alligator eggs from Lake Apopka, Lake Griffin, and Lake Okeechobee, Florida. Environ Monit and Assess. 16, 277-285. Heppel SA, Denslow ND, Folmar LC, Sullivan CV (1995). Universal assay of vitellogenin as a biomarker for environmental estrogens. Environ Health Perspect. 103, 9-15. Herbst LH, Siconolfi-Baez L, Torelli JH, Klein PA, Kerben MJ, Schumacher IM (2003). Induction of vitellogenesis by estradiol-17 and development of enzyme-linked immunosorbant assays to quantify plasma vitellogenin levels in green turtles (Chelonia mydas). Comp Biochem Physiol B Biochem Mol Biol. 135, 551-563. Hutton JM (1986). Age determination of living Nile crocodiles from the cortical stratification of bone. Copeia 2, 332-341. Hutton JM (1987). Techniques for ageing wild crocodilians. In 'Wildlife Management: Crocodiles and Alligators'. pp. 211-216. (Surrey Beatty and Sons Pty Limited: Australia.) Irwin LK, Gray S, Oberdrster E (2001). Vitellogenin induction in the painted turtle, Chrysemys picta, as a biomarker of exposure to environmental levels of estradiol. Aquat Toxicol. 55, 49-60. Iwase H and Hotta K (1993). Release of o-linked glycoprotein glycans by endo-alpha-N-acetylgalactosaminidase. Methods Mol Biol. 14, 151-159. James AM and Oliver JH Jr. (1997). Purification and partial characterization of vitellin from the black-legged tick, Ixodes scapularis. Insect Biochem Mol Biol. 27, 639-649.

PAGE 79

69 Joanen T and McNease L (1989). Ecology and physiology of nesting and early development of the American alligator. Am Zool. 29, 989-998. Joanen T and McNease LL (1980). Reproductive biology of the American alligator in south-west Louisiana. In 'Reproductive Biology and Diseases of Captive Reptiles'. (JB Murphy and JT Collins, Eds.), 153-159. Kawahara A, Sato K, Amano M (1983). Regulation of protein synthesis by estradiol 17 beta, dexamethasone and insulin in primary cultured Xenopus hepatocytes. Exp Cell Res. 148, 423-436. Kernaghan NJ, Monck E, Weiser C, Gross TS (2002). Characterization and manipulation of sex steroids and vitellogenin in freshwater mussels. Society of Environmental Toxixology and Chemistry 23rd Annual Meeting in North America, Salt Lake City, Utah. November 16-20, 2002, 263. Kushlan JA and Jacobson T (1990). Environmental variability and the reproductive success of everglades alligators. J Herpetol. 24, 176-184. Laemmeli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lance VA (1989). Reproductive cycle of the American alligator. Am Zool. 29, 999-1018. Lance VA (2003). Alligator physiology and life history: the importance of temperature. Exp Gerontol. 38, 801-805. Lance VA, Joanen T, McNease L (1983). Selenium, vitamin E, and trace elements in the plasma of wild and farm reared alligators during the reproductive cycle. Can J Zool. 61, 1744-1751. Lance VA and Lauren D (1984). Circadian variation in plasma corticosterone in the American alligator, Alligator mississippiensis and the effects of ACTH injections. Gen Comp Endocrinol. 54, 1-7. LeClerq F, Schnek AG, Braunitzer G, Stangl A, Schrank B (1981). Direct reciprocal allosteric interaction of oxygen and hydrogen carbonate sequence of the haemoglobins of caiman (Caiman crocodylus), the Nile crocodile (Crocodylus niloticus) and the Mississippi crocodile (Alligator mississippiensis). Z Physiol Chem. 1151-1158. Le Guellec K, Lawless K, Valotaire Y, Kress M, Tenniswood M (1988). Vitellogenin gene expression in male rainbow trout (Salmo gairdneri). Gen Comp Endocrinol. 71, 359-371. Lim EH, Lam TJ, Ding JL (1997). Direct submission accession # T31095. NCBI database.

PAGE 80

70 Marburger JE, Johnson WE, Gross TS, Douglas DR, Di J (2002). Residual organochlorine pesticides in soils and fish from wetland restoration areas in Central Florida, USA. Wetlands. 22, 705-711. Masson GR. (1995). "Environmental influences on reproductive potential, clutch viability and embryonic mortality of the American alligator in Florida. Gainesville, Florida, University of Florida. Matter JM, McMurry CS, Anthony AB, Dickerson RL (1998). Development and implementation of endocrine biomarkers of exposure and effects in American alligators (Alligator mississippiensis). Chemosphere. 37, 1905-1914. Morales MH, Baerga-Santini C, Cordero-Lopez N (1996). Synthesis of vitellogenin polypeptides and deposit of yolk proteins in Anolis pulchellus. Comp Biochem Physiol B Biochem Mol Biol. 114, 225-231. Morales MH, Pagn SM, Gmez Y (2002). Immunodissection of yolk lipovitellin (LV1) demonstrates the existence of different LV1-domains and suggests a complex family of vitellogenin genes in the lizard Anolis pulchellus. Comp Biochem Physiol B Biochem Mol Biol. 131, 339-348. Morales MH and Sanchez EJ (1996). Changes in vitellogenin expression during captivity-induced stress in a tropical anole. Gen Comp Endocrinol. 103, 209-219. Mouchel N, Trichet V, Betz A, Le Pennec JP, Wolff J (1996). Characterization of vitellogenin from rainbow trout (Oncorhynchus mykiss). Gene. 174, 59-64. Murakami H and Nakai M (2001). Direct submission accession # BAB79591. NCBI database. Nilsson NO, Stralfors P, Fredrikson G, Belfrage P (1980). Regulation of adipose tissue lipolysis: effects of noradrenaline and insulin on phosphorylation of hormone-sensitive lipase and onlipolysis in intact rat adipocytes. FEBS Lett. 111, 125-130. Olivecrona T, Bengtsson-Olivecrona G, Ostergaard P, Lui G, Chevreuil O, Hultin M (1993). New aspects on heparin and lipoprotein metabolism. Haemostasi.s. 23, 150-160. Palmer B and Guillette LJ Jr. (1992). Alligators provide evidence for the evolution of an archosaurian mode of oviparity. Biol Reprod. 46, 39-47. Palmer BD and Palmer SK (1995). Vitellogenin induction by xenobiotic estrogens in the red-eared turtle and African clawed frog. Environ Health Perspect. 103, 19-25. Peabody FE (1961). Annual growth zones in living and fossil vertebrates. J Morphology. 108, 11-62.

PAGE 81

71 Perutz MF, Bauer C, Gros G, LeClerq F, Vandecassarie C, Schnek AG, Brainitzer G, Friday AE, Josey KA (1981). Allosteric regulation of crocodilian haemoglobin. Nature London. 291, 682-684. Pruneta V, Autran D, Ponsin G, Marcais C, Duvillard L, Verges B, Berthezene F, Moulin P (2001). Ex vivo measurement of lipoprotein lipase-dependent very low density lipoprotein (VLDL)-triglyceride hydrolysis in human VLDL: An alternative to the postheparin assay of lipoprotein lipase activity? J Clin Endocrinol Metab. 86, 797-803. Rauschenberger RH, Wiebe JJ, Buckland JE, Smith JT, Seplveda MS, Gross TS. (2003) Achieving environmentally relevant organochlorine pesticide concentrations in eggs through maternal exposure in Alligator mississippiensis. Mar Environ Res. (In Press). Romano M, Rosanova P, Anteo C, Limatola E (2002). Lipovitellins and phosvitins of the fertilized eggs during embryo growth in the oviporous lizard Podarcis sicula. Mol Reprod Dev. 63, 341-348. Rosanova P, Romano M, Marciano R, Anteo C, Limatola E (2002). Vitellogenin precursors in the liver of the oviporous lizard, Podarcis sicula. Mol Reprod Dev. 63, 349-354. Roubal WT, Lomax DP, Willis ML, Johnson LL (1997). Purification and partial characterization of English sole (Pleuronectes vetulus) vitellogenin. Comp Biochem Physiol B Biochem Mol Biol. 118, 613-622. Ryffel GU (1978). Synthesis of vitellogenin, an attractive model for investigating hormone0induced gene activation. Mol Cell Endocrinol. 12, 237-246. Seplveda M, Johnson WE, Higman JC, Denslow ND, Schoeb TR, Gross TS (2002). An evaluation of biomarkers of reproductive function and potential contaminant effects in Florida largemouth bass (Micropterus salmoides floridanus ) sampled from the St. Johns River. Sci Total Environ. 289, 133-144. Sierra-Santoyo A, Hernndez M, Albores A, Cebrin ME (2000). Sex-dependent regulation of hepatic cytochrome P-450 by DDT. Toxicol Sci. 54, 81-87. Sumpter JP and Jobling S (1995). Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment. Environ Health Perspect. 103, 173-178. Szkudinski MW, Thotakura NR, Tropea JE, Grossman M, Weintraub BD (1995). Asparagine-linked oligosaccharide structures determine clearance and organ distribution of pituitary and recombinant thyrotropin. Endocrinology. 136, 3325-3330. Talent LG, Dumont JN, Bantle JA, Janz DM, Talent SG (2002). Evaluation of western fence lizards (Sceloporus occidentalis) and eastern fence lizards (Sceloporus undulatus) as laboratory reptile models for toxicological investigations. Environ Toxicol Chem. 21, 899-905.

PAGE 82

72 Tarentino AL and Plummer TH Jr. (1994). Enzymatic deglycosylation of asparagine-linked glycans: purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum. Methods Enzymol. 230, 44-57. Uchida Y, Tsukada Y, Sugimori T (1979). Enzymatic properties of neurominidases from Arthrobacter ureafaciens. J Biochem (Tokyo). 86, 1573-1585. Uribe MCA and Guillette LJ Jr. (2000). OOgenesis and ovarian histology of the American alligator Alligator mississippiensis. J Morphol. 245, 225-240. Vliet K (1989). Social displays of the American alligator (Alligator mississippiensis). Am Zool. 29, 1019-1031. Wahli W, Dawid IB, Ryffel GU, Weber R (1981). Vitellogenesis and the vitellogenin gene family. Science 212, 298-304. Wahli W, Martinez E, Corthesy B, Cardinaux JR (1989). Cisand trans-acting elements of the estrogen-regulated vitellogenin gene B1 of Xenopus laevis. J Steroid Biochem. 34, 17-32. Walker P, Brown-Luedi M, Germond JE, Wahli W, Meijlink FC, van het Schip AD, Roelink H, Gruber M, Ab G (1983). Sequence homologies within the 5' end region of the estrogen-controlled vitellogenin gene in Xenopus and chicken. EMBO J. 2, 2271-2279. Walker P, Germond JE, Brown-Luedi M, Givel F, Wahli W (1984). Sequence homologies in the region preceding the transcription initiation site of the liver estrogen-responsive vitellogenin and apo-VLDLII genes. Nucleic Acids Res. 12, 8611-8626. Wang H, Yan T, Tan JT, Gong Z (2000). A zebrafish vitellogenin gene (vg3) encodes a novel vitellogenin without a phosvitin domain and may represent a primitive vertebrate vitellogenin gene. Gene. 256, 303-310. Wang SY and Williams DL (1982). Biosynthesis of the vitellogenins. Identification and characterization of nonphosphorylated precursors to avian vitell9ogenin I and vitellogeninII. J Biol Chem. 257, 3837-3846. Wink C and Elsey RM (1986). Changes in femoral bone morphology during egg-lying in Alligator mississippiensis. J Morphol. 189, 183-188. Wood JM, Woodward AR, Humphrey SR, Hines TC (1985). Night counts as an index of American alligator population trends. Wildl Soc Bull. 13, 262-272. Woodruff AR and Moore CT. (1989) Statewide alligator surveys. Final report. Tallahassee, Florida, Florida Game and Freshwater Fish Commission. Woodward AR, Jennings ML, Percival HF, Moore CT (1993). Low clutch viability of American alligators on Lake Apopka. Fl Sci. 56, 52-63.

PAGE 83

73 Woodward AR, Moore CT, Delany MF (1992). "Experimental alligator harvest". Final Report. Florida Game and Fresh Water Fish Commission. Yamamura J, Adachi T, Aoki N, Nakajima H, Nakamura R, Matsuda T (1995). Precursor-product relationship between chicken vitellogenin and the yolk protein: the 40 kDa yolk plasma glycoprotein is derived from the C-terminal cystein-rich domain of vitellogenin II. Biochim Biophys Acta 1244, 384-394. Yoon S (2002). Direct submission accession # BAC20186. NCBI database. Yoshitome S, Nakamura H, Nakajo N, Okamoto K, Sugimoto I, Kohara H, Kitayama K, Igarashi K, Ito S, Sagata N, Hashimoto E (2003). Mr 25,000 protein, a substrate for protein serine/threonine kinases, is identified as a part of Xenopus laevis vitellogenin B1. Dev Growth Differ. 45, 283-294.

PAGE 84

BIOGRAPHICAL SKETCH Eileen K. Monck was born June 6, 1962 in Bronx, New York. After graduating from New Britain High School in 1980, she explored different career possibilities before enrolling at Central Connecticut State University. She graduated in 1989, with a Bachelor of Science degree in biology and secondary education (with minors in chemistry and general science). In 1989 she began working at the University of Florida as a research technician, where she developed a desire to further her education. In 1999 she began her graduate work in environmental toxicology, which she expanded to reproductive endocrinology of the American alligator. She will graduate in December 2003 with a Master of Science degree. Eileen will continue her work with alligators, under the continued supervision of Dr. Timothy Gross at the United States Geological Survey in Gainesville, Florida. Through her college career, Eileen has been a wife, and a mother to three children; and has enjoyed exposing her children to all of the fascinating educational opportunities her career has to offer. She has often been involved in bringing science into classrooms at many age levels, and looks forward to many more opportunities to do so. 74


Permanent Link: http://ufdc.ufl.edu/UFE0002851/00001

Material Information

Title: Developing a Noninvasive Method for Assessing Reproductive Status and Characterizing Gender-Specific Plasma Proteins in the American Alligator (Alligator mississippiensis)
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0002851:00001

Permanent Link: http://ufdc.ufl.edu/UFE0002851/00001

Material Information

Title: Developing a Noninvasive Method for Assessing Reproductive Status and Characterizing Gender-Specific Plasma Proteins in the American Alligator (Alligator mississippiensis)
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0002851:00001


This item has the following downloads:


Full Text












DEVELOPING A NONINVASIVE METHOD FOR ASSESSING REPRODUCTIVE
STATUS AND CHARACTERIZING GENDER- SPECIFIC PLASMA PROTEINS IN
THE AMERICAN ALLIGATOR (Alligator mississippiensis)















By

EILEEN K. MONCK


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2003


































Copyright 2003

by

Eileen K. Monck

































I dedicate this work to my family for their love and support, without which I could not
have been successful in achieving my goals.
















ACKNOWLEDGMENTS

I would like to extend my appreciation to Dr. Timothy Gross and my other

committee members, Dr. Maria Sepulveda and Dr. Evan Gallagher, for their guidance

and encouragement throughout my studies. I would especially like to thank Dr.

Sepulveda for her tutelage in the preparation of this thesis. Special thanks go to all

members of Dr. Gross's lab for making this study a success.

I would like to extend my gratitude to Dr. Nancy Denslow and the ICBR Protein

Chemistry Core Facility staff at the University of Florida. They provided an accurate and

timely analysis of my samples. Also, I would like to acknowledge the agencies

responsible for funding this project: National Institute of Environmental Health Sciences

Superfund Project #P42 ES07375; Chlorinated Pesticides and Developmental Mortality

in Wildlife, and a partial grant from The American Chemical Council to Timothy S.

Gross and Christopher J. Borgert.

















TABLE OF CONTENTS
Page

A CK N O W LED G M EN TS ................................................................... .................... iv

LIST O F TA BLES ................................................ ....... ..... ............ ............... .... vii

L IST O F FIG U R E S .............................................. ................................................. viii

ABSTRACT......... ......................... .... ...................... ix

CHAPTER

1 IN TRO D U CTIO N ......... .................................................. .................... 1

Alligator Reproductive Anatomy and Physiology.......................... ...................2
Age/Size of Sexual Maturity and Sex Ratios .................................................2
Reproductive Behavior............................ ....... ......... .................... 3
Reproductive Endocrinology..............................................5
Female Reproductive Histology and Anatomy ............................. ............ 6
Vitellogenin as a Reproductive Biomarker of Endocrine Disruption..........................9
Background and Significance ............................................. ............................... 15
Study O objectives .................. ................ .... .........................................16

2 ASSESSMENT OF REPRODUCTIVE STATUS IN FEMALE AMERICAN
ALLIGATORS ......... ....... .... ........................... .......... 17

M materials and M methods ....................................... ........... ........ .................... 19
S tu d y S ite s ................................................... ..........................................1 9
A n im a ls ................................................... ....................................................... 2 0
Plasm a Sam ples .................. ......... ... ........................... ... ................. 21
Female specific protein determination ........................... .........................21
Circulating Hormone Concentrations.....................................................23
N ecrop sies ............................................................2 5
R e su lts ................................ .................................................................2 6
D isc u ssio n .............................................................................................................. 3 5

3 IDENTIFICATION AND CHARACTERIZATION OF HIGH MOLECULAR
WEIGHT FEMALE SPECIFIC PLASMA PROTEIN BANDS.............................39

M materials and M ethods ........................................................................ 41
S tu d y S ites ................................................................. ...... ............. 4 1










A n im als ..................... .................................................................. 4 1
Female Specific Protein Determination........................................41
Enzym e D igests .................. .. ... ... ..... ......... ......... .............. ........ ........ ... 44
Anti-Phospho-serine, -tyrosine, -threonine Western Blot Analysis ..................46
Alkaline Labile Phospholipid (ALP) Analysis .................................................47
A m ino-acid Sequencing ........................................................ .................... 48
R e su lts .................................................................................................................... 5 0
D isc u ssio n .............................................................................................................. 5 2

4 CONCLUSIONS AND FUTURE DIRECTIONS ...................................................62

REFER EN CE LIST ......... ...... .............................................................................65

BIOGRAPHICAL SKETCH ................................................................ ...................74
















LIST OF TABLES


Table Page

1-1 Stages of folliculogenesis. ............................................... .................................. 8

2-1 Mean standard error of body measurements. .......................................................29

2-2 Mean standard error of the mean for reproductive measurements........................30

3-1 Amino acid sequence alignment resulting from BLAST search..............................55
















LIST OF FIGURES


Figure ae

2-1 Map of the Oklawaha River Basin, Florida ........................ ..............................31

2-2 Map showing location of Rockefeller State Wildlife Refuge...................................32

2-3 Sodium Dodecal Sulfate Polyacrylamide Gel Electrophoresis Analysis of plasma...33

2-4 Photographic documentation of reproductive tracts................................................33

2-5 Follicular frequency distribution in right and left ovaries.......................................34

3-1 Sodium Dodecal Sulfate Polyacrylamide Gel Electrophoresis Analysis of plasma...56

3-2 Glycosylation analysis of plasma samples ........................ ...........................57

3-3 Deglycosylation analysis of alligator plasma ......................................................58

3-4 Alkaline Labile Phosphate analysis of plasma proteins ..........................................59

3-5 Phospholipase digestion of alligator plasma. .......................................................60

3-6 Western blot analysis of phosphorylated proteins in alligator plasma.......................61















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

DEVELOPING A NONINVASIVE METHOD FOR ASSESSING REPRODUCTIVE
STATUS AND CHARACTERIZING GENDER- SPECIFIC PLASMA PROTEINS IN
THE AMERICAN ALLIGATOR (Alligator mississippiensis)

By

Eileen K. Monck

December 2003

Chair: Timothy S. Gross
Major Department: Physiological Sciences

Organochlorine pesticides (OCPs) in Florida lakes have been associated with

decreased egg hatchability and increased developmental mortality in American Alligators

(Alligator mississippiensis). Although concentrations of specific OCPs in yolk do not

correlate with egg hatchability and hatchling survivability, a complex mixture of OCPs

may decrease egg and embryo quality by altering maternal reproductive function.

Vitellogenin (Vtg), a follicular precursor protein has long been described as a biomarker

of endocrine disruption in other oviparous species. However, there is no documentation

in the literature of a definitive test for identifying and measuring Vtg in this species. This

is because of the inter-species variability in the amino acid sequence of this protein and

thus the low cross reactivity of commercially available antibodies. To aid in the

development of such an assay, Vtg proteins in plasma were identified and characterized

from 10 adult female alligators collected during the peak follicular period (from Lake

Griffin, FL and Rockefeller State Wildlife Refuge, LA). Two sites were chosen in an









effort to reduce site-specific bias. In addition, these sites were chosen for the ecological

significance and environmental concerns associated with alligators in these geographical

locations.

Our study was designed to develop a qualitative method for identifying follicular

females and for assessing their reproductive status; and to identify and characterize

specific plasma proteins likely to be Vtg.

Sodium dodecal sulfate-polyacrylimide gel electrophoresis (SDS-Page) analysis of

plasma revealed three prominent protein bands unique to follicular females at -250 to

450 kilo-daltons (similar to published molecular weights for Vtg or Vtg metabolites from

other oviparous species). Further characterization of these proteins revealed that they

were highly glycosylated and contained several phosphoserine amino acids. These

proteins were isolated by SDS-Page and then confirmed by protein sequencing to have

substantial homology with published Vtg sequences from other species. These data are

critical for future development of an alligator specific Vtg assay. Such an assay could be

used to further identify a possible mechanism for reproductive failures experienced by

alligator populations in contaminant-impacted environments.

Anatomical evaluations of reproductive status validated the plasma protein

screening protocol. There was a 1:1 correlation for vitellogenic females exhibiting

plasma proteins in the 250 to 450 kDa range. This correlation provided significant

evidence that this is an acceptable method for discerning Vtg animals from non-Vtg

animals. Each animal that had the three highly expressed plasma proteins also had a

larger number of follicles in the 21 mm to 25 mm or > 26 mm size classifications.
















CHAPTER 1
INTRODUCTION

Heightened awareness of endocrine disruption (ED) in wildlife is expanding to a

concern for humans as well. Many studies have helped to elucidate factors that may lead

to the current state of environmental ED. These studies have focused on several

environmental contaminants and their potential effects on many species of fish, birds,

amphibians, and reptiles. While these studies have been useful in identifying several

potential mechanisms of action for ED in these species, they have not completely fulfilled

the purpose of representing the long-term impact on the health of the entire ecosystem.

An animal model that could serve such a purpose needs to have a reasonable longevity to

age-of-sexual-maturity ratio, and an upper-level predatory status in the food chain. Most

bird, fish, amphibian, and reptilian species do not meet these criteria because they are

sexually mature relatively soon after birth; and while they may be predators, they do not

hold a very high place in the food chain. American alligators (Alligator mississippiensis)

on the other hand, are upper-level predators, have an average life span of 50 years, and

reach sexual maturity at 10 to 12 years. Their oviparous nature coupled with relatively

long egg incubations make them an excellent model for studying reptilian embryonic

development. Comprehensive embryonic studies have been conducted in normal

alligator populations (including palatogenesis and hemoglobin amino-acid sequence

development) (Densmore 1981, Le Clercq et al. 1981, Perutz et al. 1981, Ferguson 1985).

For these and other reasons, the alligator is becoming a popular model









for studying ED in the environment; and subsequently for making some preliminary

predictions for potential concerns for human exposure. Because of all of these attributes,

we chose the American alligator as our model for the development of biomarkers to

assess ED. The following work describes the development of some of these techniques.

Alligator Reproductive Anatomy and Physiology

Much of the initial information obtained on the American alligator reproductive

cycle in the southeastern United States comes from studies conducted on both wild and

captive populations by various investigators. Their compiled findings are summarized,

beginning with sexual maturity and ending with female reproductive anatomy. Our

primary focus was on Florida and Louisiana studies in order to compliment this study.

These data serve as a general overview of what is known about alligator reproduction.

Age/Size of Sexual Maturity and Sex Ratios

There is some discrepancy as to when a wild alligator reaches sexual maturity.

The general consensus is that sexual maturity depends on size, which depends on

environmental factors (e.g., temperature and food availability) that affect growth rates.

However, other factors may influence sexual maturity (such as age, genetic differences

between populations, and population densities) (Ferguson 1985). While there are

published methods using bone annual growth zones for aging (Peabody 1961, Hutton

1986), they are not applicable to females because the bone remodeling females undergo

during egg shell formation (Wink and Elsey 1986). Some general guidelines: Louisiana

males and females both reach sexual maturity at 1.8 mo or 9 to 10 y (Joanen and

McNease 1980); North Carolina males at 1.8 mo or 15 y, and females at 1.8 mo or 18 y

(Ferguson 1985); and in southern Florida at 12 to 14 y (Dalrymple 1996). Some believe

that age/size is not the only determining factor; that social order is also important. For









example, larger, dominant males (longer than 2.7 m) are more likely to breed (Joanen and

McNease 1980). These guidelines have been determined through survey analyses and

may have intrinsic errors due to sampling and/or difficulties assessing age. Captive

animals would seem to be an excellent control for these errors. However, animals reared

in captivity develop variable growth patterns due to differences in environmental factors

such as food quality and availability. This coupled with variations in mating behaviors,

which develop from being in a constrained environment, leave these animals outside the

norm and therefore unsuitable for calculating an average age/size for sexual maturity.

Sex ratios in wild populations have received some attention. They are calculated

from survey data which again can have intrinsic errors due to sampling error and/or

animal movement during the breeding season. Ferguson and Joanen (1982, 1983) tried to

approach this issue from the hatchling perspective in a 4-year study; and found

approximately five females to every male. However, open-water surveys show very

different ratios, depending on the month of sampling. These variations are probably due

to adult females remaining in the marsh, which is their preferred habitat (Ferguson 1985).

Reproductive Behavior

Courtship and mating. Typical mating rituals involve bellowing, head slapping,

and snout and head rubbing (Garrick and Lang 1977, Joanen and McNease 1989, Vliet

1989), culminating with copulation and subsequent nest building and egg deposition.

Specific timing of the breeding season can vary slightly depending on the geographical

location. However, the following data summarized by Ferguson (1985) serve as a

suitable model (similar time frames were confirmed by Guillette et al. 1997 in their

Florida study). These time points are based on air temperature (which was believed to be

the driving force); not on the length of day (note temperatures in parentheses at each time









point) for this particular study. However, there is no conclusive proof that temperature is

the only trigger involved. Many investigators believe it is a combination of factors.

In Louisiana, both females and males begin moving to deep open-water courtship

areas in the early part of March (13 C) (Ferguson 1985). Light bellowing by males (a

guttural noise made to attract a mate) is heard throughout the month of April and elevates

to mild bellowing by the end of April (21 C) which lasts until the middle of May (24 C)

when intense bellowing begins. During this entire time, females develop ova in the

ovaries; while male spermatogenesis begins in the middle of May and lasts approximately

4 weeks. Copulation begins around the third week of May and continues through the

second week of June when spermatogenesis is at its peak. Females then move to

shallower waters, and nest construction begins. Nest construction (see next section for a

description) is complete and eggs are deposited by the end of June (27 to 28 C). The

female remains to tend and protect the nest, until the next spring, when hatchlings are

ready to leave the nest. Males and non breeding females on the other hand, move to deep

open water for the remainder of the season, returning to their winter habitat by the middle

of October (22 C).

Nest construction. The female is solely responsible for building the nest. It

consists of twigs, mud, and other debris that is indigenous to the area. For example,

Florida nests consist mainly of saw grass, mud, and cotton tail grass. Typically an

experienced female will build a mounded den-type nest that has a tunnel-like entrance.

She will guard this entrance until she hears the hatchlings chirping (still inside the egg)

approximately 65 days past laying (at which time she will uncover the nest and begin

tending to the hatchlings). The average number of eggs per nest is 38 in Louisiana






5


(Joanen and McNease 1989) and 42 to 45 in Florida (Woodward et al. 1993, Masson

1995, Guillette et al. 1997). However, there are some areas in Florida that have less eggs

per nest such as Orange Lake = 33 eggs; Paynes Prairie = 34 eggs (Woodward et al.

1992) and Everglades National Park = 30 eggs (Kushlan and Jacobson 1990). Nests are

usually located near the edge of a marshy area just above the water line. This can be a

problem in times of draught that are followed by heavy rains because many nests can be

flooded out; as was the case in Orange Lake, FL the year this study was conducted

(unpublished data).

Reproductive Endocrinology

The male reproductive season begins in early spring after an increase in

circulating testosterone (T) concentrations (which peak at approximately 90 ng/mL in

April/May) (Lance 1983, 1984). This is concurrent with the production of mature sperm

that are then stored in the seminiferous tubes.

Females also have a T surge that occurs simultaneous to an increase in

17p-estradiol (E2); however the peak is less than 1/10f that of mature males (Lance,

1983). This surge of a predominantly male hormone in females is not surprising, since T

is the precursor for E2.

There is some discrepancy between the Louisiana and the Florida studies as to the

onset of the reproductive season in females. In Louisiana, Joanen and McNease (1980)

and Lance (1989) found that the alligator reproductive season begins in early spring with

an increase in circulating E2 concentrations, which peak in April at approximately 700

pg/mL. Guillette et al. (1997) determined that Florida females appear to have a bi-phasic

cycle beginning in the fall. This fall phase of increased E2 concentrations return to

summer concentrations (200 pg/mL) sometime between November and February,









however, there was no sampling during this time frame. Subsequently there is a second

increase in E2 concentrations beginning in February peaking at approximately 600 pg/mL

in April/May (Guillette et al. 1997). This second rise in E2 causes the follicles to increase

in diameter from 5 mm to 40-45 mm by late May to early June. It is unclear whether

these differences in E2 cycling between the Louisiana and Florida studies is due to

geographical variations or if it is just due to the Florida study including more time points

(Guillette and Milnes 2001). Vitellogenesis is actively going on during this time of

elevated E2 concentrations, and Guillette et al. (1997) discussed the possibility that the

fall increase in E2 concentration served to produce an initial wave of large follicles that

would in turn provide more circulating E2 which is needed for rapid oviductal growth.

The spring E2 concentrations decrease rapidly following ovulation in June.

Subsequent to the decline in plasma E2 concentrations, there is a rise in plasma

progesterone (P) concentrations beginning in April and peaking at 5-6 ng/mL in June.

This elevated concentration of plasma P continues circulating until oviposition and the

beginning of luteolysis in June/July when the P concentrations decrease to 1-2 ng/mL

(Guillette et al. 1997). Lance (1989) found that corpora lutea granulosa cells stained

positively for 3p-hydroxysteroid dehydrogenase-isomerase (3P-HSD) which is the

enzyme responsible for the synthesis of P. It is possible that plasma P produced in the

corpora lutea aids in maintaining gravidity as it does in other species (Guillette and

Milnes 2001).

Female Reproductive Histology and Anatomy

There is a right and left side to the reproductive tract; the right being the larger of

the two. However both sides are simultaneously involved during each breeding season.









Follicles are formed and nurtured in both the right and left ovaries and passed

through the oviducts, the conduits (with various functional zones) which extend to the

exterior of the body through a single vaginal opening. Folliculogenesis has been

described by Uribe and Guillette (2000) as being a series of stages which are summarized

in Table 1-1. Uribe and Guillette (2000) concluded that based on their histological

findings, stages I-VI compared to those of other reptiles, while stages VII-IX more

closely resembled birds. Other features which were similar to birds include ovarian

lacunae and smooth muscle bundles surrounding the follicles. However, there were some

characteristics which were unlike birds or other reptiles such as: yolk morphology

(animal and vegetal pole differences); yolk platelet structure; and theca morphology.

While this staging system has proved invaluable in evaluating the progress of

folliculogenesis, late-stage variations are difficult to interpret. This is mainly due to the

awkwardness of sectioning a 40 mm follicle for histological evaluations; they are very

large with very little support tissue (Guillette and Milnes 2001).

The anatomy, and functionality of the oviduct in the American alligator is more

similar to birds rather than to other reptilians. However, in contrast to birds which

completely finish one egg before ovulating the next, alligators exhibit a more

simultaneous ovulation and shelling of the entire clutch which is similar to reptilians

(Guillette and Milnes 2001).









Table 1-1: Stages of folliculogenesis.


Stage Oocyte Diameter Characteristics
(mean *SE)
Stage I: Previtellogenesis 42.8 ,6.6 m Nucleus contains chromatin in
diplotene stage of meiotic prophase I.
Thick chromosomes visible. One
nucleolus. Squamous cells begin to
surround oocyte.
Stage II: Previtellogenesis 73.8 ,6.9 m Nucleus contains lampbrush
chromosomes and one nucleolus.
Stage III: Previtellogenesis 267.3 *43.3 m Nucleus contains lampbrush
chromosomes and multiple nucleoli.
Squamous cells completely surround
oocyte; monolayer is referred to as
granulosa.
Stage IV: Previtellogenesis 486.7 *70.1 m Zona pellucida at periphery of oocyte.
Granulosa cells are cuboidal
containing a nucleus. Theca has
developed, comprised offibroblasts.
Stage V: Previtellogenesis 1.2 0.9 mm Zona pellucida is considerably
thicker, consisting of two layers; an
inner striated layer and an outer
hyaline band.
Stage VI: Vitellogenesis 3.1 0.9 mm Peripheral granules and centralized
vacuoles in ooplasm. Theca has
sinuses.
Stage VII: Vitellogenesis 4.5 *1.6 mm Granules and vacuoles have increased
greatly in numbers. Vacuoles are
much larger (up to 25 m), some
containing yolk platelets.
Stage VIII: Vitellogenesis 6.8 *3.4 mm Regional animal and vegetal poles
clearly visable. Zona pellucida 18-20
m and have well defined radiata and
hyaline layers. Theca contains blood
vessels, collagen fibers, and flattened
lacunae.
Stage IX: Vitellogenesis 19.4 *5.9 mm Ooplasm is filled with large (90 m)
yolk platelets.
Stage X: Vitellogenesis 38.8 *2.4 mm Yolk platelets continue to grow (160
m). Theca thickens to 180-200 m),
containing muscle cells as well.
Source: Summarized from Uribe and Guillette (2000).









The oviduct of the American alligator has been described in some detail (Palmer

and Guillette 1992, Buhi et al. 1999). It has been divided into seven distinct regions each

serving different purposes in preparing the mature follicle for deposition as an egg:

* The uppermost section, the anterior infundibulum, functions to receive the mature
follicle.
* The posterior infundibullum and the uterine tube are muscular with mucosal folds
and believed to function in albumen secretion.
* The utero-tubal junction is a transparent non-muscular, non-glandular section
which connects the uterine tube to the anterior uterus.
* The anterior and posterior uterus is the site of eggshell membrane formation and
eggshell calcification, respectively.
* Finally, the posterior uterus connects to the vagina where the egg exits the body.

Vitellogenin as a Reproductive Biomarker of Endocrine Disruption

Vitellogenin (Vtg) has been classified as a hormonally controlled precursor

protein to several of the yolk proteins found in oviparous eggs (Ryffel 1978). Once liver

Vtg production is stimulated by circulating E2, it is post-translationally modified and

circulated to the blood capillaries surrounding the follicular theca and transferred to the

developing oocytes by diffusion from the follicular theca and subsequent pinocytosis by

the oocytes (Wahli et al. 1981). Once in the oocytes, Vtg is proteolytically cleaved into

lipovitellin and phosvitin; however the number of cleavage products is not known for

most species (Ryffel 1978, Wahli et al. 1981). Characteristically, it is a highly

glycosylated phospho-lipoprotein. The molecular weight ranges from -150 to 600 kilo-

daltons (Kd) depending on the species (Heppel et al. 1995, Brown et al. 1997, Allner et

al. 1999, Brion et al. 2000). For example, in the African clawed-frog (Xenopus laevis), it

occurs in the form of a dimer consisting of two 200 Kd polypeptides (Wahli et al. 1981),

whereas in the Kemp's Ridley sea turtle (Lepidochelys kempi) the predominant Vtg

protein appears at 200 Kd (Heck et al. 1997). Similarly, the isoelectric point (pI), the pH









at which the net charge of the protein is zero, ranges from 6 to 7 depending on the

species (Kawahara et al. 1983, James and Oliver 1997, Roubel et al. 1997). These

characteristics were used collectively in the design of this study to optimize the chances

of correctly identifying and characterizing Vtg in the American alligator.

Since Vtg is a maternally derived protein that is utilized by the embryo as a

nutritional source, it is possible that any deviation or disruption of the pathway may alter

embryo development. The full extent to which the developing embryo uses Vtg is not

clearly understood, but if it could act as a carrier protein for xenobiotic chemicals, then it

would stand to reason that enzymes used by the embryo to metabolize those chemicals

could be turned on and the activity up-regulated. Metabolism is a complex process in

that enzymes are developed to control more than one event. For example cytochrome

P450 enzymes are instrumental in xenobiotics metabolism as well as steroid metabolism

(Ertl et al. 1999, Sierra-Santoyo et al. 2000). With this in mind it is possible that other

events in the developing embryo could be affected by this exposure. There are several

potential pathways and functions to explore but first there must be a definitive method for

identifying and characterizing Vtg in the species being studied. This has been done for

fish and birds, but there is limited information in amphibians other than Xenopus and

reptiles.

Vtg has been proposed as a biomarker of exposure to endocrine disrupting

chemicals (EDC) in oviparous species (Sumpter and Jobling 1995). The rationale behind

using Vtg as a biomarker stems from extensive research using the African clawed frog

and the chicken (Gallus domesticus) as models for investigating E2 induced Vtg gene

activation (Ryffel 1978). Studies on the Japenese medaka (Oryzzas latipes) revealed that









Vtg may be induced in males by E2 and EDC to produce Vtg at a level previously

determined to be indicative of a reproductive female (Gronen et al. 1999). More recently,

Vtg has been investigated in Florida as a biomarker of potential endocrine disrupting

effects in largemouth bass (Micropterus salmoldes) (Bowman et al. 2002, Sep* iveda et

al. 2002). Numerous studies have been conducted in other species to identify and

characterize this class of proteins (Wang and Williams 1982, Wahli et al. 1989, Hartling

et al. 1997) and while there has been some work done in reptilian species such as lizards

and turtles (Baerga-Santini and Morales 1991, Brown et al. 1997, Morales et al. 2002,

Romano et al. 2002), there is very little reported for the crocodilians (Guillette et al.

1997). The reptilians which have been investigated most conclusively in this respect are

turtles and lizards. The following is a brief summary of the most recent studies

published.

In the past 3 y there have been three comprehensive studies which have been

essential in advancing turtle Vtg research to the point where quantitative assays are now

possible. Duggan et al. (2001) analyzed plasma from the freshwater painted turtle

(Chrysemys picta) in a seasonal study to fully characterize seasonal lipid transport in this

species. They concluded that in this species, lipids and proteins control seasonal ovarian

growth probably under hormonal control. These authors provide a detailed protocol for

monitoring plasma lipids in turtles which utilized several techniques including the well

known gravimetric method for total lipids as well as enzyme-linked immunosorbant

assay (ELISA) methods for individual lipid components. Irwin et al. (2001) designed a

study to analyze the potential effects of xenoestrogens present in cattle farm pond water

on Vtg induction in the painted turtle. The rationale behind this study was that the









manure runoff into the ponds could be carrying metabolized (glucoronide-conjugated)

hormones which bacteria in the water could subsequently cleave into active steroids.

These in turn could potentially induce the turtles and fish (male and female) that

inhabited the ponds to increase hepatic Vtg production, therefore altering their

reproductive cycles. They used an ELISA method designed to measure Vtg in both

males and females from the affected ponds and compared them to a control site. They

found that water concentrations of xenoestrogens in the water were sufficient to induce

Vtg production in females but not in males. Herbst et al. (2003) recently published a

comprehensive study designed to analyze the Vtg protein sequence in green turtles

(Chelonza mydas) and compared it to published sequences from other species (tuatara

[Sphenodon punctatus], chicken, and frog). They found that the n-terminal sequence

obtained from 15 cycles of Edman degradation protein sequencing was not an exact

match to anything in the National Center for Biotechnology Information (NCBI) or the

Expressed Sequence Tags (EST) databases. The sequence however, had 73% homology

with that of the tuatara (Brown et al. 1997). They then purified plasma Vtg to produce

polyclonal and monoclonal antibodies to egg yolk granules which was reactive to green

turtle Vtg in both ELISA and Western blot analyses.

Lizard Vtg research has advanced from mere identification and MW

determination (Carevali et al. 1991, Baerga-Santini and Hernandez de Morales 1991) to

time course analysis beginning with hepatic induction and ending with deposition in the

developing follicle (Morales et al. 1996). Another interesting study conducted by

Morales and Sanchez (1996) continued on their time course studies and investigated the

effects of captivity on anole (Anolis pulchellus) Vtg production and subsequent follicular









deposition. They found that long term captivity stress induced cessation of Vtg

production and circulation could be alleviated by low level E2 hormone replacement

therapy within 72 -96 h.

Talent et al. (2002) and Brasfield et al. (2002) both published studies advocating

lizards as a potential reptilian model for ecotoxicological risk assessments. Talent et al.

(2002) designed an egg injection study which revealed that 17c-ethinylestradiol (an

estrogenic chemical) caused male embryo feminization by impeding the development of

secondary sex characteristics. Brasfield et al. (2002) designed a study to aid in the

development of a protocol which could potentially be used as a quantitave tool for

monitoring Vtg in western fence lizards (Sceloporus occidentals). This study utilized a

direct Vtg ELISA method and compared it to an indirect plasma alkaline-labile phosphate

(ALP) method previously used in invertebrates (Kernaghan et al. 2002) and fish (Gagne

et al. 1998, 2000, 2001) as an indirect measure of Vtg. They concluded that there was a

high correlation between the two methods and that the ALP method could be a suitable

measure of plasma Vtg in fence lizards.

Rosanova et al. (2002) contributed invaluable data to the Vtg field by identifying

the MW and location in two liver subcellular fractions of several Vtg precursor proteins

in the oviparous lizard (Podarcls slcula). This study utilized Western blot analysis to

identify two proteins (84 and 70 kDa) located in the rough endoplasmic reticulum (RER)

and four proteins (180, 150, 60, and 50 kDa) located in the smooth microsomal fraction.

Romano et al. (2002) conducted a time course study on the oviparous lizard

(Podarcis slcula) which followed the fate of lipovitellins and phosvitins previously

identified in egg yolks over a course of 44 days from ovoposition. There were two









lipovitellins at 110 and 116 kDa that remained constant in the yolk throughout the 44 day

incubation. The phosvitin profile underwent various changes throughout the 44 day

incubation periods; on day one there were four proteins detected at 50, 45, 29, and 14

kDa; on day 10 post ovoposition, the 29 kDa phosvitin was missing but a new one was

detected at 6.5 kDa; on day 18, only two phosvitins were detected at 14 and 6.5 kDa; and

finally at day 44, only the 6.5 kDa phosvitin was detected. This suggested that there was

a continuous degredation of the phosphorylated proteins in the egg yolk over the course

of incubation. The interpretation of this degradation was that the embryo needed to be

supplied with amino acids and smaller proteins during its embryonic growth. This study

confirmed a need for assays that are capable of tracking these specific phosphorylated

proteins or protein fractions throughout the time course which extends from egg

production in the adult female liver through the developmental period of the embryo if

we are to begin to elucidate the effects that contaminants (which may cause oxidative

damage and subsequent dephosphorylation) may have on the reproductive success of

these and other reptilian species.

Heppel et al. (1995) attempted to develop a universal Vtg ELISA that would be

reactive with plasma Vtg from several species including a snake and tuatara. The authors

found that Vtg was only two to three times higher in vitellogenic females when compared

to males (lizards and snakes), while the fish female reactivity was three to 10 times

higher than the males depending on the species.

Previous efforts to examine Vtg proteins in crocodilians have been qualitative or

semi-quantitative. Matter et al. (1998) attempted to modify the method developed by

Palmer and Palmer (1995)to quantify Vtg in hatchling alligators. Briefly, they









performed Western blot analyses of hatchling plasma utilizing a rabbit anti-Vtg antibody

which was raised against red-eared turtle Vtg. They were unable to detect an induction of

plasma Vtg in hatchlings; however this was probably due to their young age coupled with

continued lipovitellin and phosvitin contribution from their yolk sac. Brown et al. (1997)

utilized an antibody raised against tuatara (Sphenodon punctatus) in a western blot

analysis of adult female alligator plasma successfully recognizing a specific protein at

-220 kDa which they presumed to be Vtg. However this was not expanded upon, since

the subject of their study was the tuatara. There has not been a quantitative assay

published to date that is sensitive and specific for crocodilians. The current study was

designed to characterize and isolate Vtg in the American alligator as a critical step toward

the development of a quantitative assay for this species.

Background and Significance

The American alligator was placed on the United States endangered species list in

1967 (Groonbridge 1987). At that time, it was an acceptable practice to allow unlimited

harvesting of animals for the sale of meat, skins, and trinkets such as teeth, claws, and

skutes. It was even acceptable to harvest hatchlings and sell them as "pets". Alligator

populations appeared to be diminishing, therefore monitoring of the species was begun to

determine the extent of the threat for extinction. Now after years of monitoring, experts

agree the species has made an important recovery and is no longer in danger of extinction

(Wood et al. 1985, Woodruff et al. 1989). However, the monitoring program that was

established opened a new venue for environmental research and alligators became a

popular model for contaminant studies in the southeastern United States due to their place

in the food chain, their longevity, and therefore their potential for bioaccumulation of

xenobiotics (Hall and Henry 1992, Crain and Guilette 1998). In fact, it has been









proposed that many contaminants alligators are exposed to may be EDs (Gross et al.

1994, Guillette et al. 1994, Crain et al. 1997, Guillette and Gunderson 2001, Guillette et

al. 2002). Alligator research in this area was originally conducted on eggs to determine a

potential relationship between contaminants and their effects on reproductive success.

However, to date no clear relationship has been established between the level of

contaminants found in the eggs and embryo survival (Heinz et al. 1991). Therefore, an

increasing number of researchers have begun looking at the adult female for a better

understanding of mechanisms) behind altered reproductive success.

Due to the many factors that contribute to growth and maturity in this species

such as temperature, population density, and food availability and quality (Hutton 1987),

it is nearly impossible to determine if an adult female is reproductively active and will lay

eggs in a particular year based on anatomical size alone. This coupled with permit

limitations has led to the need for developing a non-invasive tool for evaluating the

reproductive status of adult female alligators. The development of such a tool was the

primary goal of this work. A secondary goal was to begin to isolate and characterize

plasma Vtg from this species. This is of importance because it will aid future studies in

elucidating a potential mode(s) of action of EDC.

Study Objectives

The objectives of the present study were to

1. Develop a qualitative method for identifying follicular females, and to

2. Identify and characterize female specific plasma proteins likely to be Vtg.















CHAPTER 2
ASSESSMENT OF REPRODUCTIVE STATUS IN FEMALE AMERICAN
ALLIGATORS

Alligators are a popular model for reptilian contaminant studies due to their

predatory place in the food chain, their longevity, and therefore their potential for

bioaccumulation of contaminants (Hall and Henry 1992, Crain and Guilette 1998). It has

been proposed that many of the contaminants that alligators are exposed to may be ED's

(Gross et al. 1994, Guillette et al. 1994, Crain et al. 1997, Guillette and Gunderson 2001,

Guillette et al. 2002). Contaminant research in this species was originally conducted on

alligator eggs to determine a potential relationship between contaminants and their effects

on reproductive success. So far, no clear relationship has been established between the

level of contaminants found in the eggs and embryo survival (Heinz et al. 1991).

Therefore, an increasing number of researchers have begun looking at the adult female

for a better understanding of the mechanisms) behind altered reproductive success.

Due to the many factors that contribute to growth and maturity in this species,

such as temperature, population density, and food availability and quality (Hutton 1987),

it is nearly impossible to determine if an adult female is reproductively active and will lay

eggs in a particular year based on anatomical size alone. This, coupled with permit

limitations (as in Florida) has led to the need for developing a non-invasive tool for

evaluating the reproductive status of adult female alligators. To date there is no such

protocol published for alligators. This study was designed to develop a novel plasma









protein assay which could be utilized for the prediction of reproductive status in adult

female alligators.

Vitellogenin (Vtg) protein is produced in the livers of reproductive female

alligators, circulated through the blood, and subsequently deposited in the developing

follicles. Along with being a reliable predictor of gravid females, it has also been

proposed as a biomarker of exposure to EDC in oviparous species (Sumpter and Jobling

1995). However, to date, there has not been a quantitative assay published that is

sensitive and specific for crocodilians. Heppel et al. (1995) attempted to develop a

universal Vtg ELISA that would be reactive with plasma Vtg from several species

including reptiles, but found that it was not sensitive enough (for reptiles) to be

considered a reliable assay. Matter et al. (1998) attempted to modify the Western blot

developed by Palmer and Palmer (1995) to be used as a quantitative Vtg assay in

hatchling alligators. They were unable to detect an induction of plasma Vtg in the

hatchlings, however this was probably due to their young age coupled with continued

lipovitellin and phosvitin contribution from the yolk sac. Another factor to consider is

the potential non-specific reactivity of the antibody with non-Vtg proteins. The current

study was therefore designed with the intent that the data obtained herein could be used

to further the efforts in developing such an assay that would be sensitive and specific for

alligators.

The primary objective of this study was to screen several free-ranging alligator

females and develop a reproducible method for evaluating reproductive status. Animals

were screened initially for the presence of highly expressed plasma proteins specific to

adult females in the 250 to 350 kDa. This is the predicted MW range for Vtg in other









oviparous species (Heppel et al. 1995, Brown et al. 1997, Allner et al. 1999, Brion et al.

2000). This is a relatively non-invasive procedure which should decrease the incidents of

sacrificing animals that don't fit the study's criteria. A secondary objective of this study

was to develop a standardized necropsy protocol which could be used to quantitatively

assess the reproductive tract and thus be a tool for use in comparative studies.

Materials and Methods

Study Sites

Two sites were chosen in an effort to reduce site specific bias from being

introduced into the individual experiments. Each site was chosen for its significance to

the ecological and environmental concerns surrounding alligators in their respective

geographical locations (see following sections for relevance of chosen sites).

Lake Griffin, Florida. Florida Lakes in the Ocklawaha River Basin have been

the subject of environmental concern for the past few decades (Benton and Douglas 1994,

Marburger et al. 2002). In the 1980's, Lake Apopka's alligator population declined

noticeably suggesting a potential association with organochlorine pesticides (OCP)

(Guilette et al. 1995). There were several point sources responsible for OCP

contamination in Lake Apopka and subsequently the entire basin (Benton and Douglas

1994, Marburger et al. 2002). Lake Griffin, located downstream of Lake Apopka

(Figure. 2-1), has moderate to elevated OCP concentrations in alligator egg yolks and

decreased egg viability (Rauschenberger et al. 2003).

Rockefeller State Wildlife Refuge, Louisiana. Rockefeller Wildlife refuge was

donated to the State of Louisiana in 1920, and it is comprised of 76,042 acres (this is

down from the original 80,000 acres due to erosion) which border the Gulf of Mexico

(Figure. 2-2). The Deed of Donation mandated that the land be maintained as a wildlife









refuge, and that there would be no public or commercial fishing or trapping. In 1983

there was an amendment to allow sport fishing and commercial trapping for the purpose

of generating revenue for education and public health. This was amended again in 1987

ceasing the surplus revenue (Louisiana Department of Wildlife and Fisheries). Since

then, it has been maintained as a refuge and it is staffed by a team of scientists,

conservation officers, and of course a maintenance crew. The research conducted at

Rockefeller has been instrumental in many of the advancements made in alligator

ranching and physiology. There is limited access allowed to the public with regulations

that are strictly enforced. This refuge has become popular as a reference site for many

studies due to its low levels of soil contaminant concentrations (Elsey et al. 1999, Davis

et al. 2001, Cobb et al. 2002) and the reduced level of stress to wildlife.

Animals

Adult female alligators (1.8 2.1 m) were captured by noose according to IACUC

guidelines. Captures were coordinated such that animals from each site were at

equivalent points in their reproductive seasons: Rockefeller animals were captured in mid

April and Lake Griffin animals were captured in early May (these dates were chosen to

target animals which would be in the late vitellogenic (V) stage of their reproductive

cycle). These time points were confirmed to be similar when eggs were collected and

staged later in the season: Rockefeller embryos were collected June 14t and staged at

day 7-12 on June 29t, and Lake Griffin embryos were collected and staged at day 12 on

July 1". Animals were held in a moist cool enclosure until they were screened for the

study criteria described below. Those meeting the criteria were held for sacrifice and

those which did not meet the criteria were returned to their place of capture and released.









Plasma Samples

Blood (10 mL) was drawn from the occipital sinus into a heparinized syringe and

transferred to heparinized tubes. The blood was set on ice until it could be centrifuged at

1000 rpm for 20 min in a Beckman J6-HC centrifuge to separate plasma from the cellular

fraction. Once separated the plasma was snap-frozen in 1 mL aliquots and stored at

-800C.

Female specific protein determination

Sodium dodecal sulfate polyacrylamide gel elctrophoresis analysis was performed

according to the method described by Laemmeli (1970) to screen plasma for the presence

of female specific proteins in the predicted MW range (-250 to 450 kDa) for Vtg. A

predetermined criteria was set to categorize the plasma profiles in the 250 to 450 kDa

range as being (1) "highly vitellogenic" if the female specific protein bands were at least

two to three times more intense than that of a positive control female or (2) "weak to non-

vitellogenic" if the female specific proteins were less intense than those of the control

female or not present at all. These intensities were measured utilizing one individual's

gross visual judgment due to the nature of the field set-up and lack of availability of a

scanning densitometer. The positive control female used for these and subsequent

experiments had been implanted with a 180 day time release pellet containing 20 mg of

E2 in September 2001. Subsequently, plasma was drawn in December 2001 and

preserved according to the protocol described previously (unpublished data, Gross et al.

2001).

Protein extractions. All chemicals utilized in this section were purchased from

Sigma-Aldrich Company Corp., St Louis. MO, USA. Plasma samples (100 L) were

clarified by spinning at 10,000 rpm for 5 min in an Eppendorfmicrocentrifuge (to









remove cellular components). A surfactant extraction buffer (containing a protease

inhibitor cocktail made up of 4-(2-aminoethyl)benzenesulfonyl fluoride [AEBSF];

ethylenediaminetetraacetate [EDTA]; Bestatin, L-trans-3-Carboxyoxiran-2-carbonyl-L-

leucylagmatine [E-64]; Leupeptin; and Aprotinin) was applied to plasma samples to

liberate and denature proteins. This was prepared from a 10x extraction buffer which

consisted of 500 mM Tris-HCl pH 8.0, 100 mM EDTA pH 8.0, 5% Triton-X 100, 2%

sodium dodecylsulfate (SDS), 5% sodium deoxycholate (DOC), and 20 L/mL protease

inhibitor cocktail. The 10x buffer was added to 90 L of clarified plasma to give a lx

final concentration. Samples were kept on ice during extraction to minimize degradation.

Protein assay. Chemicals, pre-cast gels, protein standards, and equipment

utilized in this and subsequent electrophoresis sections were purchased from Bio-Rad

Laboratories, Hercules, CA, USA. Extracted protein samples were quantified according

to Bradford (1976) using the Bio-Rad Protein Assay kit. A 1:100 dilution of each plasma

sample was quantified by measurement against a bovine serum albumin (BSA, supplied

in kit) standard curve ranging from 0 to 40 g/mL total protein. The micro protein assay

was performed by pipetting 160 L of each sample dilution and standard into a 96 well

plate in triplicate. Subsequently 40 L of G250 protein dye reagent (supplied in kit) was

added to each well. Plates were incubated at room temperature for 5 min and

subsequently read on a Dynex MRX Microtiter Plate Reader at 595 nm. Dynex

Revelation software was used to develop a standard curve and extrapolate sample protein

concentrations.

Sample preparation. For each animal, 20 g total plasma protein, was prepared

by adding sample reducing buffer containing 12.5% 0.5 M Tris-HCL pH 6.8; 25%









glycerol; 2% SDS; 5% P-mercaptoethanol; and 1.25% bromophenol at 2 3 times the

sample volume and boiling for 1 min to denature the protein.

Electrophoresis. The denatured protein samples were loaded onto 7.5%

acrylamide denaturing gels for maximum high MW separation and reasonable band

definition. A lx running buffer (25 mM Tris base, 250 mM glycine, and 0.1% SDS) was

used to perform electrophoresis on a Bio-Rad Mini-gel II apparatus powered by a Bio-

Rad Power Pac 200. Molecular weights were confirmed by comparison to denatured

MW standards which were run simultaneously with samples on each gel. Subsequent to

electrophoresis, gels were fixed and stained with coomassie brilliant blue (CBB) protein

staining solution composed of 40% methanol, 10% acetic acid, and 1% CBB overnight at

room temperature with gentle agitation. The next day they were washed in several

changes of de-stain (40% methanol and 10% acetic acid) to remove unbound stain and

equilibrated in double deionized water (ddH20). The gels were then dried between two

pieces of cellophane sheets in a Bio-Rad Air Dryer for preservation and subsequent

documentation on a Bio-Rad GS-800 densitometer.

Data analysis. SDS-Page gels were analyzed and qualitatively graded for

intensity of female specific bands in the 250 to 450 kDa MW range. These intensities

were measured utilizing one individual's gross visual judgment due to the nature of the

field set-up and lack of availability of a scanning densitometer. Only those animals that

presented intensity in all female specific bands (when compared to a male plasma pool

and the positive control female plasma [described previously]) were considered to be

highly folliculogenic and subsequently chosen for sacrifice (see Figure. 2-5).

Circulating Hormone Concentrations

Plasma samples were analyzed by a standard radioimmunoassay (RIA) procedure









to determine circulating E2 and T. This is a competitive binding assay set up to allow

competition between the animal's plasma hormone and a radiolabeled standard hormone

for the binding site of a protein antibody. The following is a summary of the method

from Giroux (1998).

Sample extractions. For each hormone assay, plasma (50 ) was extracted

twice with 4 mL of ethyl ether in duplicate (two tubes, each containing 50 i of plasma

from each animal) for each hormone assay. Tubes were then vortexed for 1 min and then

incubated in a methanol/dry ice bath for 3 to 4 min to precipitate (freeze) the aqueous

fraction of the plasma. The ether/lipophilic plasma fraction was poured into a 100 mm

glass tube and placed on a Labconco evaporator for 10 to 15 min. This procedure was

repeated using the same tubes, thereby concentrating the two extractions together.

Hormone assays. Standard curves and samples were prepared as follows. Total

count tube (TC): 350 L phosphate buffered saline with 1% gelatin and 0.01% sodium

azide (PBSGA) was added to 100 L radioactive label to determine the upper limit of the

radioactivity in the assay; non-specific binding tube (NSB): 350 L phosphate buffer was

added to 100 L radioactive label to measure its reaction with the antibody; zero binding

tube (BO), 250 L phosphate buffer was combined with 100 L radioactive label and

100 L antibody to determine maximum binding of the unlabeled Ab-Ag complex;

standard curve tubes, 200 L phosphate buffer was combined with 100 L radioactive

label and 100 L antibody and 50 L known steroid in eight tubes of increasing

concentrations from 0 pg/mL to 20,000 pg/mL; extracted sample pellets, 250 L

phosphate buffer was combined with 100 L radioactive label and 100 L antibody

specific for either E2 or T. All tubes were incubated for 24 hours at 40C. The next day









250 L charcoal dextran was added to all tubes except the TC tubes and subsequently

centrifuged for 10 min at 3000 rpm and 40C in a Beckman J6-HC centrifuge to remove

the unbound antibody. For each tube, 0.4 mL of the supernatant was taken off and added

to 4 mL of Scintiverse scintillation fluid (Fisher Scientific, Fairlawn, NJ, USA) in

scintillation vials (United Laboratory Plastics, St. Louis, MO, USA). Samples were

counted on a Packard Tri-Carb scintillation counter (model 1600CA). Unknown samples

were quantified against the standard curve using the Beckman EIA/RIA Immunofit.

Necropsies

Twenty adult female alligators were screened at each site. Of the 20 animals from

each site, 10 highly V and 3-5 weak to non-vitellogenic (NV) animals (for contrast) were

sacrificed and necropsied. Anatomical reproductive tract evaluations were performed

according to a standardized protocol (Table 2-1 and 2-2). Linear measurements (total

length: tip of nose to tip of tail; snout-vent length; head length; and tail girth which was

measured just behind the vent) were performed using a centimeter tape. Weight was

determined by suspending the animal from a kilogram scale which was attached to a fork

lift. Animals were sacrificed by cervical dislocation and double pithing. Subsequently,

necropsies were performed according to the following protocol saving appropriate tissues

for further analysis. The abdominal cavity was exposed by making two transverse cuts:

one at the vent and one just below the chest cavity. Subsequently a longitudinal cut was

made on one side at the transition between the dorsal and ventral side. The outer skin and

fat layer was then filleted away from the abdominal membrane and the flap retracted.

The abdominal cavity was further exposed by cutting away the rib cage and through the

tough outer membrane. Once inside the cavity, organs were dissected out, weighed, and









measured. The liver was weighed on a gram scale, and color and condition noted. The

entire reproductive tract was removed from both the right and left sides.

Photo-documentation was performed using a centimeter ruler for scale. The oviduct and

ovaries were separated, weighed and measured (oviductal diameters were taken in the

center of each anatomical section [defined in Chapter 1], lengths were not recorded due

to expected inaccuracies subsequent to stretching and straightening). All follicles greater

than 5 mm were counted, weighed, and measured using a gram scale and digital calipers.

Health and reproductive parameters were evaluated using the following formulas:

* Condition factor; K = 100 x (weight (g)/length (cm)3)
* Hepatic Somatic index; HSI= 100 x (liver wt/body wt -liver wt)
* Gonadal somatic index; GSI = 100 x (gonad wt/body wt -gonad wt)

Statistics were run for mean, SEM, equality of variance, and ANOVA (for

multiple groups with sites) or T-tests (for individual means between sites) when

appropriate using the Minitab statistical package.

Results

SDS-Page analyses revealed three bands in the Vtg MW range (-250, 350, and

450 kDa) that were present in higher concentrations in follicular animals (indicated by

brackets in Figure. 2-3). These results correlated well (10 animals out of 10) with

anatomical evaluations (Figure 2-4 panels A & B). Each animal from both sites that

presented intense plasma protein bands in the above mentioned MW range (Fig 2-3 panel

A) also presented a highly follicular (a greater number of large [>20 mm] follicles)

reproductive tract (Fig 2-4 panel A & B). Conversely, each animal from both sites that

presented weak to non-existent plasma protein bands in the above mentioned MW range

(Fig 2-3 panel B) also presented a weakly follicular (a greater number of small [<20 mm]









follicles) reproductive tract (Fig 2-4 panel C & D).

Table 2-1 summarizes the average anatomical evaluations of all animals from

both sites that were necropsied. Overall, Lake Griffin (LG) V and NV females were

significantly larger (snout-vent length, head length, tail girth, and weight) when

compared to Rockefeller (R) animals. Lake Griffin V females had a significantly higher

condition factor when compared to R V animals but this was not true for the NV females

when sites were compared. While there were significant differences noted between sites

for the previously mentioned lengths (indicated in parenthesis), the total lengths were not

significant. However, there was an overall trend for the LG animals to be longer than the

R animals. The Rockefeller V animals had a significantly higher hepatosomatic index

(HSI) when compared to LG V animals but this was not true for the NV females. There

were no significant differences for any of the previously mentioned parameters noted

when the V animals were compared to the NV animals within sites.

Table 2-2 summarizes the average reproductive evaluations of all animals from

both sites that were necropsied. Lake Griffin V animals had significantly larger oviductal

weights and diameters when compared to R V animals. However, there were no

significant differences noted for the oviductal measurements or ovarian weights for the

NV females. The LG V females however had a significantly higher GSI compared to the

R V animals while there was no significant difference between sites for the NV animals.

The average numbers of follicles (overall totals and size class totals) were

summarized in Table 2-2. There were no significant differences in the overall number of

follicles when LG V animals are compared to R V animals. However, there were

differences in the distribution of these follicles in the different size classes. For instance,









LG V animals had significantly more 5-10 mm follicles in the right ovary and also

contained follicles in the > 26 mm category, which was absent in the R V females, and R

V animals had significantly more 16-20 mm follicles in both ovaries when compared to

the LG animals. The distribution and frequency of the follicular size classes for each of

the V animals are summarized for the two sites separately in Fig 2-5. These graphs

reiterate the results obtained from the averages determined in Table 2-2. Overall, the LG

V animals had a large number of predominantly > 26 mm follicles (Fig 2-5 panels C &

D); while the R V animals had a larger number 16-22 mm and 21-25 mm follicles (Fig

2-5, panels A and B). In summary, when comparing reproductive tract measurements of

V animals across sites, LG animals had significantly larger tracts containing a greater

number of large follicles (> 26 mm).

However, when comparing NV animals across sites (for all of the above

reproductive measurements), there were only two significant differences noted: LG NV

animals had significantly more 5-10 mm follicles, whereas R NV animals had

significantly more 16-20 mm follicles.

When the V animals were compared to the NV animals within sites (Table 2-2),

the following significant differences were noted for the previously described reproductive

measurements: LG V animals had significantly larger and heavier oviducts and ovaries

than the LG NV animals, and for both sites V animals had higher GSI compared to NV

animals. In addition, the R V animals had significantly more follicles overall.

Conversely, there was no significant difference noted for the LG animals due to high

variability in the NV animals, however, the trend indicated that there were more total

follicles in the LG V animals. When the follicle size classes were compared, R V









animals had significantly more 5-10 mm, 11-15 mm and 21-25 mm follicles than the R

NV animals. The LG V animals had significantly more follicles 21-25 and > 26 mm

follicles, while the LG NV animals did not have any follicles of these size classes.

The average plasma E2 concentrations were 432 39 ng/mL and 571 73 ng/mL

for R and LG V animals, respectively. The circulating T concentrations were 219 119

ng/mL and 279 66 ng/mL for R and LG V animals, respectively. There was no

significant difference between these values for either hormone across sites. There was no

hormone analysis performed on the NV animals. This was due to technical difficulties

that arose after the V animals had been analyzed.

As stated previously, there was a direct correlation (10 out 10 animals for each

site) between the three female specific bands noted on the SDS-Page analysis of the

plasma and the physical appearance of the reproductive tract (Figures 2-3 and 2-4).

Table 2-1. Mean standard error of body measurements.

Rockefeller Lake Griffin
V NV V NV
Total length (cm) 232 7 224 + 10 245 + 8 247 + 4
Snout-vent length (cm) 120 4 118 5 133 3a 154 26
Head length (cm) 371 36 1 41 + la 40 0.4a
Tail girth (cm) 56 2 53 3 66 3a 66 1 a
Weight (kg) 38 4 36 7 59 6a 56 4
Condition factor 0.3 + 0.01 0.3 + 0.02 0.4 0.02 a 0.4 + 0.01
Hepatosomatic Index 1.3 + 0.01a 1.1 + 0.2 0.9 + 0.04 0.9 + 0.1

a Indicates a significant difference between sites (p < 0.05).
(vitellogenic (V) sample size: n = 10, for each site; non-vitellogenic (NV) sample size: n
= 3, for each site)













Table 2-2. Mean standard error of the mean for reproductive measurements.


Rockefeller Lake Griffin
V NV V NV


Oviduct diameter (mm)
Upper
Middle
Lower


212a
191
382ab


19+1ab
181 b
394ab


Oviduct weight (g/kg body weight)

Ovary weight (g/kg body weight)


Gonadal-somatic index:

Total number of follicles


5-10 mm
11-15 mm
16-20 mm
21-25 mm
> 26 mm


Plasma Estrogen (pg/mL)
Plasma Testosterone (pg/mL)
a Indicates a significant difference


4.80.5b 5.00.5b


4.40.5


1.70.5 1.80.9


6.90.7ab 7.30.8ab


4.90.5 3.00.8 3.20.9 9.21.2ab 8.70.9ab


0.40.1


294b


71 b
41 b
102a
113b
0


343

41
21
21
63b
203ab

57173
27966


4324
219119


between sites (p


34+3

52
31
10.5
63 b
193ab


2.10.7

1.00.2

0.90.1


2.20.7

1.10.1


1311 2013


154a
55
0
0
0


0.05). Indicates a significant difference between V and NV within sites (p


0.05. Missing samples from two animals, therefore no SEM. N/A: there was no hormone data available for NV animals.
(vitellogenic (V) sample size: n=10, for each site; non-vitellogenic (NV) sample size: n=3, for each site)

















































Figure 2-1 Map of the Oklawaha River Basin, Florida Arrow indicates Lake Grnfin






































ROCKEFELLER


WILDLIFE -
REFUGE

Figure 2-2. Map showing location of Rockefeller State Wildlife Refuge. Only part of the
refuge is shown where the study took place (indicated by arrow).









MW R1


R2 R3
. 'i


MW **4 **R4 R5
r~fr


G2 G3


G4


150 -l


Figure 2-3 SDS-PAGE analysis of plasma samples from adult female alligator plasma
Brackets indicate expected molecular weight (MW) range for V proteins 9 lane
contains plasma from an E2 induced control female J lane contains plasma from a
control adult male alligator pool Analysis normalized to total protein loaded
A) Vitellogemc (V) (n 3 from Lake Griffin [G] and Rockefeller [R]) B) Non-
vitellogemc (NV) (n= 2 from Lake Griffin [G] and Rockefeller [R])


Figure 2-4 Photographic documentation of reproductive tracts of representative animals
from each site A) V Rockefeller female B) V Lake Gnffin female C)NVpre-
ovulatory Rockefeller female D) NV pre-ovulatory Lake Gnffin female














R1 to R10 Left Ovary

50
40
30 -
20 O
10 _
0

Total 5-10 11-15 16-20 21-25 26+


C G1 to G10 Left Ovary

50
401
40 -
30 -

20

Total 5-10 11 -15 16-20 21 -25 26+


Figure 2-5: Follicular frequency distribution in right and left ovaries.
Y axis: The number of follicles in each size classification.


Total 5-10 11-15 16-20 21-25 26+


Each bar represents one female alligator.


X axis: Total (total number of follicles for each animal), followed by each size classification (measured in mm).


R1 to R10 Right Ovary

50
40 -
30
20
10

Total 5-10 11-15 16-20 21 -25 26+


G1 to G10 Right Ovary


Lj IN









Discussion

The three female specific proteins that were identified by this study have provided

a good starting place for investigating plasma Vtg in alligators. They are within the

predicted MW range based on information that has been published for other species, but

it must be confirmed that one or all of these proteins are truly Vtg (see Chapter 3).

Secondly, a full molecular characterization must be done to determine their origination

and subsequent fate such as follicular deposition.

The anatomical and reproductive evaluation section of this study was designed to

serve three purposes: (1) to validate the results of the plasma protein screening; (2) to

provide a comprehensive data base of anatomical and reproductive parameters of both

Vtg and non-Vtg adult females; and (3) to use the data generated to develop a

standardized protocol to be used for future evaluations of female alligators' health and

reproductive status.

The evaluation of reproductive status validated the plasma protein screening

protocol. There was a 1:1 correlation for V females exhibiting plasma proteins in the 250

to 450 kDa range. This correlation provided significant evidence that this is an

acceptable method for discerning Vtg animals from non-Vtg animals. Each animal that

had the three highly expressed plasma proteins also had a larger number of follicles in the

21 to 25 mm or > 26mm size classifications. There was also very little inter-animal

variability in the oviductal diameters. This coupled with the low variability in their sizes

(within sites) confers the likelihood that they were equivalent in age and gravida (number

of times that they had reproduced).

Alligator reproductive anatomy has been described comprehensively in the

literature dating back as far as the 19th century. While there was no new information









acquired from this study as far as reproductive anatomy, it did provide an essential

teaching tool for this student's education in reptilian anatomy. Subsequently, another

opportunity arose in that a second investigator (Dr. Dave Rostal, Georgia Southern

University) was simultaneously performing abdominal ultrasonography on the same

animals used for this study in an effort to validate his method for use in alligators. There

was a 1:1 correlation between Dr. Rostal's results (predetermined criteria for a positive

ultrasound was detection of follicles >15 mm) and the anatomical evaluations performed

in this study. This collaboration proved to be beneficial to both groups while limiting the

needless sacrifice of additional animals.

The E2 hormone values were close to the expected average of 700 pg/mL for adult

female alligators during the latter part of the reproductive cycle for both of these

geographical locations. Testosterone values however, were well above the published

average of 90 ng/mL. A possible explanation for the discrepancy in the average T values

obtained is that the T hormone assay was lacking in sensitivity and/or specificity for

alligators. Comparing this study's E2 analysis with others in the literature showed it to be

useful in providing another parameter to evaluate the point the animals were at in their

reproductive status, however it may have been improved by also including P in the

hormone profile.

This study demonstrated that a qualitative analysis of female specific plasma

protein in alligators was a useful and predictive measure of folliculogenesis. There was

an increase in the number of follicles in LG animals which did not coincide with a higher

level of plasma E2 confirming that E2 had already peaked for the season and that these

animals were in late vitellogenesis. This also suggests that there may be a different, non-









hormonally induced pathway at work in LG animals driving them to produce a greater

number of larger follicles. Another possibility is that the hormone assay performed in

this study lacked sensitivity and/or specificity for alligator plasma. However, since the

values were comparable to those in the literature, this is not a very likely explanation.

The anatomical evaluations demonstrated that overall LG animals were

significantly larger than R animals with a higher condition factor indicating that the LG

animals probably had more body fat as well. This could be due to seasonal variations

between the two sites as females begin to mobilize fat deposits while they progress

through their reproductive season. This could be an explanation for the larger follicles

present in the LG animals. It seems to be a plausible possibility that the R animals were

going to go through the same process later in the season thereby placing them slightly

behind the LG animals in their reproductive status, however the egg staging data suggests

the opposite. The R animals were captured and subsequently eggs were collected 2-3

weeks earlier than LG animals. The equivalency which is suggested in the materials and

methods section is probably skewed because the R eggs had a longer period of time

between when they were collected and when they were set in the incubator due to

transportation. Two possible explanations for the reproductive differences between these

two sites are: the animals were at equivalent stages when they were sacrificed with R

animals having begun their season earlier; or, perhaps it is due to the fact that R females

lay smaller eggs. Rockefeller animals had a higher HSI than the LG animals suggesting

that they were in the process of increased hepatic protein production. Since Vtg is a

hepatic protein, then perhaps they were just behind the LG animals in their Vtg

production which would explain why they had smaller follicles overall. A









comprehensive time course following the production of Vtg coupled with plasma

hormones would need to be done at both of these sites to fully understand the variations

between these two sites.

Having a comparison of non-Vtg and Vtg animals within these sites provided

another piece of information vital to the study of reproductive biology. This study

reflected that 50% of the screened population at each site would go on to reproduce that

year. This compares to other studies which have found that 63 to 68% of the adult female

population reproduce in a given year. It is difficult to determine the accuracy of this

study's data since all 20 animals from each site were not sacrificed. However, based on

the three non-Vtg animals from each site that were sacrificed, it is likely that the "non-

Vtg" animals would not have gone onto reproduce that year. It is a widely accepted

supposition that crocodilian reproduction is temperature driven coupled with water level

of the nesting areas. These two factors are inherently seasonal for each geographical

location. This makes the theory of a second wave of reproductive females very unlikely.















CHAPTER 3
IDENTIFICATION AND CHARACTERIZATION OF HIGH MOLECULAR WEIGHT
FEMALE SPECIFIC PLASMA PROTEIN BANDS

Vitellogenin (Vtg) has been classified as a hormonally controlled precursor

protein to several of the yolk proteins found in oviparous eggs (Ryffel 1978). Once Vtg

production is stimulated by circulating estradiol (E2) in the liver, it is post-translationally

modified and circulated to the blood capillaries surrounding the follicular theca and

transferred to the developing oocytes by diffusion from the follicular theca and

subsequent pinocytosis by the oocytes (Wahli et al. 1981). Once in the oocytes, Vtg is

proteolytically cleaved into lipovitellin and phosvitin, however the number of cleavage

products is not definitively known and varies between species (Ryffel 1978, Wahli et al.

1981). Characteristically, it is a highly glycosylated phospho-lipoprotein. The precursor

protein (circulated through the plasma) MW ranges from -150 to 600 kilo-daltons (kDa)

depending on the species (Heppel et al. 1995, Brown et al. 1997, Allner et al. 1999, Brion

et al. 2000). For example, in the African clawed-frog (Xenopus laevis), it occurs in the

form of a dimer consisting of two 200 kDa polypeptides (Wahli et al. 1981), whereas in

the Kemp's Ridley sea turtle (Lepidochelys kempi) the predominant Vtg protein appears

at 200 kDa (Heck et al. 1997). Similarly, the isoelectric focusing point (pI) ranges from

- 6 to 7 depending on the species (Kawahara et al. 1983, James and Oliver 1997, Roubel

et al. 1997). These characteristics were used collectively in the design of this study to

optimize the chances of correctly identifying and characterizing Vtg in the American

alligator.









The most extensive characterization of Vtg is in fish, birds (mainly chickens and

quail), and amphibians (mainly the African clawed frog). Although little is known about

this protein in reptiles, this research is rapidly growing and it is gaining popularity as a

model for environmental endocrine disruption.

Since Vtg is a maternally derived protein that is utilized by the embryo as a

nutritional source, it is possible that any deviation or disruption of the pathway may alter

embryo development. Subsequently it has been proposed as a biomarker of exposure to

endocrine disrupting chemicals in oviparous species (Sumpter and Jobling 1995). The

rationale behind using Vtg as a biomarker stems from extensive research using the

African clawed frog and the chicken(Gallus domesticus) as models for investigating

estrogen induced Vtg gene activation (Ryffel 1978). Studies on the Japenese medaka

(Oryzzas latipes) revealed that Vtg may be induced in males by E2 and endocrine

disrupting chemicals to produce Vtg at a level previously determined to be indicative of a

reproductive female (Gronen et al. 1999). More recently, Vtg has been investigated in

Florida as a biomarker of potential endocrine disrupting effects in largemouth bass

(Micropterus salmoldes) (Bowman et al. 2002, Sep* iveda et al. 2002). Numerous studies

have been done in other species to identify and characterize this class of proteins (Wang

and Williams 1982, Wahli et al. 1989, Hartling et al. 1997); and while there has been

some work done in reptilian species (described above) such as lizards and turtles

(Baerga-Santini and Hernandez de Morales 1991, Brown et al. 1997, Morales et al. 2002,

Romano et al. 2002), there is very little reported for the crocodilians (Guillette et al.

1997). There has not been a quantitative assay published to date that is sensitive and

specific for crocodilians. The current study was designed to characterize and isolate Vtg









in the American alligator as a critical step toward the development of a quantitative assay

for this species.

The previous chapter identified three female specific plasma proteins in the 250 to

500 kDa MW range which were present in higher concentrations in folliculargenic

animals. Therefore the objectives of this study were to identify and characterize those

high MW plasma proteins. We tested the hypothesis that Vtg was represented by one of

these three bands.

Materials and Methods

Study Sites

Two sites (Rockefeller State Wildlife Refuge, Louisiana and Lake Griffin,

Florida), were chosen in an effort to reduce site specific bias from being introduced into

the individual experiments. Each site was chosen for its significance to the ecological

and environmental concerns surrounding alligators in their respective geographical

locations (see previous chapter for a description of sites).

Animals

Adult female alligators (1.8-2.1 m) were captured, euthanized, and necropsied

according to IACUC guidelines as described in Chapter 2. Plasma samples were

obtained and preserved as described in Chapter 2.

Female Specific Protein Determination

The following methods were performed as described in Chapter 2 with the

following modifications. Chemicals, pre-cast gels, protein standards, and equipment

utilized in this and subsequent electrophoresis sections were purchased from Bio-Rad

Laboratories, Hercules, CA, USA, or from Sigma-Aldrich Company Corp., St Louis.

MO, USA except where indicated otherwise.









Protein extractions. Plasma samples (100 L) were clarified by spinning at

10,000 rpm for 5 min in an Eppendorf microcentrifuge (to remove RBC's and WBC's).

A non-ionic surfactant extraction buffer (without inhibitor cocktail to allow for enzyme

digestions) was applied to plasma samples to liberate and denature proteins. This was

prepared from a 10x extraction buffer composed of 500 mM Tris-HCl pH 8.0, 100 mM

EDTA pH 8.0, 5% Triton-X 100 (SDS and DOC (due to their ionic nature) were not

included in this buffer to allow for proper isoelectric focusing (IEF) described in the

following section). The 10x buffer was added to 90 i of clarified plasma to give a ix

final concentration. Samples were kept on ice during extraction and alliquotted prior to

snap freezing and subsequent storage at -800C to minimize degradation.

Protein assay. Extracted protein samples were quantified according to Bradford

(1976) using the Bio-Rad Protein Assay kit previously described in Chapter 2.

Sample preparation. For each animal, 20 g total plasma protein, was prepared

by the method previously described in Chapter 2.

Electrophoresis. The denatured protein samples were then loaded onto 4 to 15%

gradient acrylamide denaturing gels for maximum high MW separation while allowing

for the capture of the entire protein profile from 250 kDa down to 25 kDa.

Electrophoresis was then performed according to the method described previously in

Chapter 2. Subsequently, gels were stained with coomassie brilliant blue for MW

determination and dried between cellophane for documentation. A second set of gels

were run simultaneously and stained for glycosylated proteins using a modified Periodic

Acid-Schiff (PAS) method.









Isoelectric focusing analysis. Semi-purified samples (prepared as follows) were

utilized to determine the pi of the three female specific proteins to allow for a cleaner

more focused analysis in lieu of conventional 2D-SDS Page analysis which can be very

complex therefore limiting the ability to discern the protein of interest. The denatured

protein samples (one animal chosen randomly from each site, extractions described

previously) were loaded onto 7.5% acrylamide denaturing gels for maximum high MW

separation (7 wells for each animal). Electrophoresis was then performed according to

the method described in Chapter 2. The three female specific bands of interest were then

excised and eluted in SDS-Page running buffer (7 slices for each band from each animal

were combined in a separate elution tube) using the Bio-Rad model 422 Electro-eluter.

This yielded 6 individual semi-purified samples; 1-250 kDa, 1-350 kDa, and 1-450 kDa

protein for each of the Rockefeller and Lake Griffin animals. These samples were then

concentrated using Centricon YM-100 spin columns (Millipore Corporation, Billerica,

MA, USA) by centrifugation at 1000 rpm for 30-60 min. Subsequently, the samples

were diluted to 2 mL in PBS and re-concentrated three times to exchange the buffer and

remove the SDS. Samples (10 ng quantified by the protein assay described previously in

Chapter 2) were combined with rehydration buffer (8 M urea, 2% 3-[(3-

Cholamidopropyl)dimethylammonio]- -propanesulfonate hydrate [CHAPS]; 40 mM

dithiothreitol [DTT]; 0.2% Bio-LyteTM 3/10 ampholyte: Bio-Rad Ready/Prep 2-D starter

kit), loaded onto pre-cast 7 cm immobilized pH gradient (IPG) strips (pH 5 to 8; Bio-

Rad), and allowed to hydrate overnight. The strips were transferred to a clean, dry

PROTEAN IEF focusing tray which was subsequently placed in the Bio-Rad PROTEAN

IEF focusing cell and allowed to focus (covered with mineral oil) using the pre-set









protocol (outlined below) determined by the length and number of strips. The strips were

then stained with IEF stain from Bio-Rad (27% Isopropanol; 10% acetic acid; 0.04%

coomassie blue R-250; 0.05% crocein scarlet) and subsequently destined with 50%

methanol and 10% acetic acid until bands were discernable. The pi was noted and

recorded.

Pre-set protocol utilized to focus the proteins in the IEF focusing cell:

* Start voltage = 0 V
* End voltage = 8,000 V
* Volt-hours = 8-10,000 V-hr
* Ramp= rapid
* Temperature = 20 C

Enzyme Digests

Plasma protein extractions described above were utilized to perform the following

enzyme digests in an effort to confirm which (if not all) of the three female specific

proteins contained phospho-lipid and sugar moieties. The digests were then analyzed by

SDS-Page according to the method described above to determine any changes in MW of

the three female specific proteins created by removing their covalently bonded groups.

Deglycosylation. The E-DEGLY kit from Sigma was utilized to completely

remove all N-linked and simple O-linked carbohydrates from the alligator plasma

proteins. The kit contains the following enzymes; PNGase F (Chryseobacternum

[Flavobacterium] meningosepticum) which cleaves all asparagine-linked complex,

hybrid, or high mannose oligosaccharides (Tarentino et al. 1994) unless a-core

fucosylated (Szkudinski et al. 1995); a-2(3,6,8,9) Neuraminidase (recombinant from

Arthrobacter ureafaciens) which cleaves all non-reducing terminal branched and

unbranched sialic acids (Uchida et al. 1979); O-Glycosidase (recombinant from









Streptococcus pneumonia) which cleaves serine or threonine-linked unsubstituted Gal-

P(1-3)-GalNAc-a- (Glasgow et al. 1977, Iwase et al. 1993); P(1-4)-Galactosidase

(recombinant from Streptococcus pneumonia) which releases only P(1-4)-linked, non-

reducing terminal galactose (Glasgow et al. 1977); and P-N-Acetylglucosaminidase

(recombinant from Streptococcus pneumonia) which cleaves all non-reducing terminal 3-

linked N-acetylglucosamine residues (Glasgow et al. 1977). For purposes of this study

the following protocol was followed under denaturing conditions. Total plasma protein

(100 g) was diluted to 30 i with deionized water (ddH20) in an Eppindorf tube, 10 i

of 5x reaction buffer, 2.5 i of denaturation solution (both supplied in kit -proprietary

ingredients), 2.5 i of Triton X-100 solution, and 1 i of each enzyme (all in one tube to

achieve complete deglycosylation) was added, mixed gently and incubated overnight at

370C. Subsequently 1/5t of this reaction was analyzed by SDS-Page on a 4-15%

gradient gel, stained with CBB, dried, and scanned for photodocumentation (all described

in chapter 2).

Phospholipase digestion. Lipoprotein lipase (LPL) is found in vivo associated

with heparin sulfate proteoglycans (HSPG) at the luminal surface of vascular

endothelium (Olivecrona et al. 1993). It is essentially responsible for hydrolyzing

triglycerides (TG) from very low density lipoprotein (VLDL) particles (Nilsson et al.

1980, Eckel 1989). Pruneta et al. (2001) isolated plasma VLDL and added exogenous

bovine LPL to monitor the TG hydrolysis. The experiment described below was a

modification of that study in that after the digestion, SDS-Page analysis was performed

instead of monitoring the kinetics of the assay. Lipoprotein lipase (Sigma) was added (6

to 7 units/10 ) to 100 g total plasma protein (diluted to 10 i with ddH20) and 90 i









lx reaction buffer (100 mM sodium phosphate, 150 mM sodium chloride, and 0.5% (v/v)

Triton X-100; pH 7.2). Subsequently 1/5t of this reaction was analyzed by SDS-Page on

a 4 15% gradient gel, stained with CBB, dried, and scanned for photo-documentation

(all described in Chapter 2).

Anti-Phospho-serine, -tyrosine, -threonine Western Blot Analysis

Plasma protein extractions described above were utilized to perform the following

Western blot analysis in an effort to confirm which (if not all) of the three female specific

proteins were phosphorylated and to identify which of the three most likely

phosphorylated amino acids they contained. Electrophoresis was performed as described

previously. Subsequently the protein was transferred to a 0.45 m nitrocellulose

membrane (Bio-Rad) for Western blot analysis. The protein transfer was accomplished

by the following protocol optimized for the Bio-Rad mini trans-blot apparatus (Bio-Rad);

gels, nitrocellulose membranes, whatman filter paper, and sponges were equilibrated for

15 minutes in transfer buffer (20% methanol in 25 mM Tris base, 250 mM glycine, and

0.01% SDS). Transfer sandwiches were then assembled in the following sequence;

sponge on black side of holder, filter paper, gel, nitrocellulose, filter paper, and sponge.

The holder was then locked and placed into the transfer module with the black side facing

the black side of transfer module. The module was placed in the electrophoresis tank

equipped with an ice block and filled with transfer buffer. Transfer proceeded at 90 volts

constant for 2.5 hrs surrounded by ice to reduce chances of protein degradation due to

overheating. Once the transfer was complete, the following immunoblot protocol was

followed; membrane was rinsed quickly with ddH20 and subsequently blocked for 1 h in

blocking buffer (5% BSA in PBS with 0.05% Tween-20 [PBS-T]); it was then incubated









with constant agitation in a UVP HB-2000 Hybrilinker hybridation oven (Tango

Technologies, Ltd., Boulder, CO, USA) in monoclonal primary antibody diluted in wash

buffer (PBS-T) overnight at room temperature (RT) at the following dilutions: mouse

anti-phosphoserine (Sigma) at 1:1000; mouse anti-phosphothreonine (Sigma) at 1:50; and

mouse anti-phosphotyrosine (Sigma) at 1:2000. The next day the membrane was washed

3 x 5 minutes in wash buffer and subsequently incubated 1 hour at RT in goat anti-mouse

IGG (alkaline phosphate conjugated) secondary antibody (Sigma) diluted to 1:30,000 in

wash buffer. A final wash of 3 x 10 minutes with was buffer and lx with ddH20 was

performed prior to color development. Detection was performed by incubating the

membrane in Western Blue Stabilized Substrate for Alkaline Phosphatase (Promega,)

until bands of interest appear at desired intensity. This is a nitro blue tetazolium (NBT)

and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) substrate which turns a purple color

when acted upon by alkaline phosphatase.

Alkaline Labile Phospholipid (ALP) Analysis

ALP analysis is an assay which has been used in some fish and invertebrate

studies as an indirect assay for plasma or hemolypmph (respectively) Vtg. It was utilized

in the current study as a secondary method to confirm the increased concentration of

phospholipid proteins in the plasma of the "highly vitellogenic" females when compared

to the "weak to non-vitellogenic" females. The method is described as follows:

Plasma (350 L) was transferred to 16 x 125 mm glass tube containing 350 L

tert-butyl methyl ether, mixed well, and incubated at room temp for 30 min (mixing every

10 min). This step extracted the lipophillic components (lipids and lipoproteins). The

tubes were centrifuged in a J6-HC centrifuge for 2min at 3,000 rpm to ensure separation.









The organic phase was transferred to a new tube and mixed with 100 L of 1 M NaOH

and incubated at RT for 60 to 90 min (mixing well every 15 min). This step freed the

alkali-labile phosphates. The tubes were centrifuged in a J6-HC centrifuge for 2 min at

3,000 rpm to ensure separation. The aqueous phase (containing the free phosphates) was

transferred to a new tube for subsequent analysis by a modified (scaled down 1:5 to be

performed in a 96 well format) phosphomolybdenum method (Phosphate Assay Kit; Sigma)

which assays for inorganic phosphorous. Each sample (15 L in triplicate) was

combined with development reagent consisting of TCA, molybdenum reagent, and Fiske-

SubbaRow reducer and was quantified by measurement against an aqueous inorganic

phosphorus standard curve (also in triplicate) ranging from 0 to 1.8 g/mL. A micro-

protein assay was performed (as described previously) on the original plasma sample and

results were utilized to report the ALP values in g of organic phosphate / mg protein.

Amino-acid Sequencing

Once the female specific proteins had been analyzed to determine that they had

characteristics of Vtg from other species (correct MW, pi, phosphorylation,

glycosylation, and phospholipid moieties), amino acid sequencing needed to be

conducted to definitively identify them as Vtg. While there are publications that have

utilized direct n-terminal sequencing by Edman degredation to identify Vtg, they have

been marginally successful in obtaining full sequences. Therefore, for the purposes of

this study internal polypeptide sequencing subsequent to enzyme digestion (of the whole

protein) was chosen. The enzyme digestions reduced the protein to smaller polypeptide

fragments thereby avoiding the covalently bonded groups that normally interfere with n-

terminal sequencing. Plasma (25 g per well) from one animal from each site chosen









randomly was electrophoresed as described previously in seven wells of a 7.5%

polyacrylamide gel (a separate gel for each animal). The gels were stained with

coomassie brilliant blue (as described previously), the three female specific bands in the

250 to 450 kDa range in each lane were excised along with a blank gel slice, same size

bands combined as one sample (keeping bands from the two animals separate; yielding 6

samples -three different size bands for each animal and 2 blank slices), and subsequently

sent to the Interdisciplinary Center for Biotechnology Research (ICBR) Protein

Sequencing Core for amino acid sequencing. The protocol performed by the Core is

summarized below.

In-gel digestion of proteins in polyacrylamide gel pieces. Each gel slice was

cut into -1 x 2 mm sections, placed into a 1.5 mL micro centrifuge tube with 150 L of

50% acetonitrile in 0.2 M ammonium bicarbonate (pH 8.9), and incubated for 30 min at

37 C. This wash buffer was removed and the wash step repeated. The gel slices were

then dried completely in a speed vacuum. Endoproteinase Asp-N enzyme (Roche

Laboratyories,) solution was added to each sample using a 1:20 w/w ratio with 50 L

0.2 M ammonium bicarbonate (pH 8.9) and incubated for 24 h at 37 C. The total volume

of the sample (gel & buffer) was estimated and 45 mM DTT was added to give a final

DTT concentration of 1 mM and subsequently incubated for 20 min at 50 C. The

samples were cooled to RT and an equal volume (to DTT volume) of 100 mM iodoacetic

acid (IAA) was added and subsequently incubated for 20 min at RT in the dark. The

supernatant was transferred to a new tube. The gel pieces were crushed and incubated for

30 min at RT with 100 L of 0.1% TFA / 60% acetonitrile. This extraction buffer was

transferred to a filter tube and extraction was repeated combining the 2nd extraction with









the first in the filter tube. Gel pieces were then discarded and the filter tube was

centrifuged for 10 -15 min at maximum speed. The speed vacuum was used to decrease

the final volume to < 150 L. This filtrate was then applied to an equilibrated Vydac

C18 ( 2.1 x 150 mm, 300 "pore size, and a 5 m particle size) reversed-phase HPLC

column at 0.15 mL/min in 95% buffer A / 15% buffer B. Elution from the column was

performed with buffer A / buffer B mixture according to the following gradient: 0-110

min (5% to 75% buffer B), and 110-120 min (75% to 85% buffer B). Elute was collected

in 1.5 ml tubes which were capped immediately and stored at 40C until sequencing was

performed.

Protein sequencing. Repeated cycles of Edman degredation chemistry was

utilized for n-terminal sequencing (on an Applied Biosystems model 494 HT Sequencer)

of the polypeptides resulting from the enzyme digestions. Briefly, this entails the

reaction of phenylisithiocyanate (PITC) with the n-terminal amino group of the

polypeptide under mildly alkaline conditions to form an n-terminal PITC adduct. This

was subsequently cleaved by anhydrous trifluoroacetic acid (TFA) yielding a

thiazolinone derivative leaving the rest of the polypeptide intact. The thiazoline-amino

acid was extracted into an organic solvent and subsequently treated with an aqueous acid

to form a more stable phenylthiohydantoin (PTH) which was later identified by gas

chromatography. Sixteen cycles were acquired with a sampling rate of 4.0 hz and

detector scale of 1.0 AUFS.

Results

Three female specific bands were again detected by SDS-Page at -250, 350, and

450 kDa (Figure 3-1). Upon isolation of the three bands (one Vtg plasma sample was









chosen randomly from each site for this procedure), the pi was found to be -7.2 for all

three bands in both samples (data not shown).

Glycosylation of the three female specific protein bands was determined by two

methods: staining of an SDS-Page gel by a modified Periodic Acid-Schiff (PAS)

method (Figure 3-2) and enzyme deglycosylation and subsequent analysis by SDS-Page

to detect a shift in the electro-mobility (Figure 3-3). The PAS staining method was

successful in identifying all three bands as being glycosylated in all 10 Vtg females from

both sites (Figure 3-2 is a representative gel showing three animals from each site). This

was further confirmed by enzyme deglycosylation of one Vtg plasma sample chosen

randomly from each site and subsequent analysis SDS-Page (Figure 3-3). However the

enzyme deglycosylation only showed an electrophoretic shift in the 250 kDa protein.

An indirect method for the quantification of phospholipids was used initially to

establish that there was a higher concentration of these lipophilic molecules in the plasma

of Vtg females when compared to non-Vtg females. There was a significantly higher

amount of phospholipid protein in the plasma samples of the Vtg females when compared

to a male plasma pool, however there was no significant difference noted when sites were

compared (Figure 3-4). These results were not strengthened by digesting one Vtg plasma

sample from each site with phospholipase and subsequent analysis by SDS-Page (Figure

3-5). There was no significant electrophoretic shift noted in the 250 to 450 kDa proteins.

This enzyme only digests phospholipid moieties, it will not digest a phosphorylated

amino acid.

Western blot analysis was used to identify which of the three bands (if not all)

were phosphorylated and which of the three most commonly phosphphorylated amino









acids did these proteins contain. The anti-phosphoserine blots revealed that the 250, 350

and the 450 kDa protein bands contained a high concentration of phosphorylated series

(Figure 3-6 Panel A). The anti-phosphotyrosine blots revealed that only the 250 kDa

band contained phosphorylated tyrosine amino acids while the anti-phosphothreonine

blots did not reveal any degree of phosphorylation in any of the three bands (Figure 3-6

Panel B and Panel C respectively). This was a third method confirming that the three

proteins in the 250 to 450 kDa range are phosphorylated.

Finally, while the sequencing project is still ongoing, preliminary results for the

250 kDa protein have revealed a 75 to 88 % homology when compared to published

chicken, frog and fish Vtg sequences (see sequence alignments in Table 3-1). This is a

small fragment resulting from the reconstruction of two out of five enzyme digest

fractions of the 250 kDa protein from the Lake Griffin animal. There are 17 residues in

this sequence with the highest confidence on residues 4-13. The sequence of this

fragment is as follows; E (Glutamine) V (Valine) G (Glycine) I (Isoleucine) R (Argenine)

A (Alanine) E (Glutamine) G (Glycine) L (Leucine) G (Glycine) X (unidentified). A

sequence homology search was performed utilizing the Basic Local Alignment Search

Tool (BLAST) which is provided through the National Center for Biotechnology

Information (NCBI) server. Of the 100 sequences that were returned in the query, 11 of

them were Vtg sequences from various species of chickens, frogs, and fish.

Discussion

This study was designed to meet the following objectives: (1) isolate and

characterize the three female specific bands that had been identified in the previous

screening study and (2) use what little is known in the literature about alligator Vtg to

prove that one or all of those three bands are or are not Vtg.









Characteristically Vtg has been proven to be a highly glycosylated phospholipid

protein in other species. The published MW weight ranges from 150 to 600 kDa

depending on the species being investigated. There have also been some lower MW

products which have been referred to as "Vtg-like". Vtg is a complex protein that

originates in the liver of oviparous vertebrates. Much of the confusion in regards to the

actual size of the protein is probably due to the fact that it undergoes extensive post-

translational processing upon transfer out of the liver as well as after it begins it's journey

through the bloodstream and then again prior to being taken up by the oocytes. The form

that shows up in an assay is dependant on many factors including the reproductive status

of the animal being tested. Vtg production and modification can be affected by hormonal

influences, diet, and other environmental factors including seasonal changes. Taking all

of this into account, the present study was designed to target the most likely candidates

and analyze them for characteristics specific to Vtg or Vtg-like proteins. Secondly,

utilize amino acid sequencing and submit this data to the available sequence banks as a

method to identify Vtg.

The initial characterization of the three female specific proteins can be

summarized as follows: (1) The 250 kDa protein is highly glycosylated and contains

several phosphorylated serine amino acids as well as some phosphorylated tyrosines.

The phospholipase digestion showed no electrophoretic mobility shift indicating that

there was very little (or no) phospholipid present. The pi is -7.2 which is in the predicted

range for Vtg. (2) The 350 kDa protein is highly glycosylated and contains several

phosphorylated serine amino acids as well as some phosphorylated tyrosines. The

phospholipase digestion did not show an electrophoretic mobility shift indicating that









there were very little (or no) phospholipid moieties present. The pi is -7.2 which is in the

predicted range for Vtg. (3) The 450 kDa protein is glycosylated but to a lesser degree

than the other two proteins. It contains several phosphorylated serine amino acids. The

phospholipase digestion showed minimal electrophoretic mobility shift indicating that

there was very little (or no) phospholipid present. The pi is -7.2 which is in the predicted

range for Vtg. Another explanation for the failed phospholipase digestion could be that

the reaction is just not sensitive enough to discern the phospholipid moieties due to the

complex nature of the Vtg protein. It is possible that these moieties are protected in the

folding of the protein which would not allow for proper digestion by the phospholipase

enzyme.

The preliminary amino acid sequencing revealed that the nine residues obtained from the

250 kDa protein have 75 to 88 % homology with published sequences from chickens,

frogs, and fish. This data coupled with the characterization described previously infers

that the 250 kDa female specific protein identified in this study is probably Vtg.

Conclusion of the sequencing project should provide sufficient evidence to confirm this

and to identify the other two female specific proteins as well.












Table 3-1: Amino acid sequence alignment resulting from BLAST search. Query represents the nine residues obtained from the 250
kDa female specific protein isolated from American alligator (Alligator mississippiensis) plasma.

% homology to Reference
Species Start Sequence End homology to Reference
query sequence
QUERY 1 EVGIRAEGL 9
Chicken (Gallus gallus) 679 E V G I R V E G L 687 88 % Walker et al., 1983
Chicken (Gallus gallus) 679 E V GIA A E G L 687 88 % Yamamura et al., 1995
African clawed frog (Xenopus laevis) 681 E I G I R GEG 688 75 % Walker et al., 1984
African clawed frog (Xenopus laevis) 681 E VA L R A E G L 689 77 % Yoshitome, 2003
Japanese whiting (Sillao aponica) 679 E V G V R A E G 686 87 % Yoon, 2002
Blue tilapia (Oreochromis aureus) 679 E V G V R T E G 686 75 % Lim et al., 1997
Rainbow Trout (Oncorhynchus mykiss) 679 E V G V R T E G 686 75 % Le Guellec et al., 1988
Rainbow trout_(Oncorhvnchus mkiss) 679 E V G V R T E G 686 75 % Mouchel et al., 1996
Zebrafish_(Danio rerio) 678 G I R A E G L 684 85 % Wang et al., 2000
Japenese medaka (Oryzias latipes) 680 E V G V R T E G 687 75 % Murakami & Nakai, 2001
CONSENSUS EVG*R* EGL


(-) Denotes missing amino acid.
(*) Denotes lack of consensus.








0 *


R1 R2 R3 G1 G2 G3

B pl,*3

can. i
La~~~ -c i


50

37 4|jSwhA..

25 left


OJ& ....... OW'-" son""":'f,


- aN


w:=, i. l- .- S.W -.:


*0ll
I----


Figure 3-1. SDS-PAGE analysis of plasma samples from three Vtg adult female
alligators. Three were from Lake Griffin [G] and three from Rockefeller [R]). Gel was
stained with coomassie brilliant blue. Brackets surround expected molecular weight
(MW) range for Vtg proteins. Y lane contains plasma from an E2 induced control
female. J' lane contains plasma from a control male pool. Analysis normalized to total
protein loaded. As was noted in Chapter 2 (Figure 2-3 panel A), there are three
prominent bands in the 250450 kDa MW range for both sites.


vv..








R I R2-5- 3qj- G2 G3

*& L *::a V L
low")* C


5(1. .....


Figure 3-2 Glycosylation analysis of plasma samples SDS-PAGE analysis of plasma
samples from three Vtg adult female alligators (three from Lake Gnffin [G] and
Rockefeller [R]) stained for glycosylation using a modified Penodic Acid-Scuhff (PAS)
method Brackets surround expected molecular weight (MW) range for Vtg proteins
lane contains plasma from an E2 induced control female $ lane contains plasma from a
control male pool Analysis normalized to total protein loaded PAS stain only stains
proteins that are glycosylated It is clear that the three bands in the 250-450 kDa range
are highly glycosylated in animals from both sites


"0 "








F Fx BSA BSAx *
k--


R Rx


G G1x MW


Figure 3-3 Deglycosylation analysis of alhgator plasma SDS-PAGE analysis of
plasma samples from two Vtg adult female alligators (one from each site) stained with
coomassie bnlliant blue One of each sample was deglycosylated by enzyme digestion
pnor to being electrophoresed Samples without enzyme are indicated by X F lanes
contain Feutin, protein positive for glycosylation BSA was included as a negative
control for glycosylation Brackets surrounds expected molecular weight (MW) range
for Vtg proteins Successful deglycosylation is identified by the electrophoretic
mobility of the protein shifting down indicating a lower MW There was only shght
deglycosylation noted in the 250 kDa protein for both the Lake Gnffin and the
Rockefeller animals


~" C











1000


750 -


500
323 is male

250 plasma value
Rock Griffin

Figure 3-4. ALP analysis of plasma proteins. ALP analysis confirming the increased
concentration of phospholipid proteins in the plasma of the vitellogenic females when
compared to the male pool (indicated by horizontal line).








MW BSA
1


BSAx


R1 R1x


-aiit irjJ


1.


i


.4@sa*J~


Figure 3-5. Phospholipase digestion of alligator plasma. SDS-PAGE analysis of plasma
samples from two Vtg adult female alligators. One from Lake Griffin [G] and one from
Rockefeller[R]) stained with coomassie brilliant blue. One of each sample was treated
with phospholipase prior to being electrophoresed. Samples without enzyme are
indicated by X. 6 lane contains plasma from a control male pool. BSA was included as
a negative control. Brackets surround expected molecular weight (MW) range for Vtg
proteins. Successful dephosphorylation would be identified by the electrophoretic
mobility of the protein shifting down indicating a lower MW. However, there was no
dephosphorylation noted for either the Lake Griffin or the Rockefeller animal.


G1x
-*fIfiij


G1


iLg(







MW B R1 G1 BSA R1


250
150
100

75 -


50

37


BSA R1 G1


BSA R1 G1


11


Figure 3-6 Western blot analysis of phosphorylated proteins in alhgator plasma
Western blot analysis analysis of plasma samples from two Vtg adult female alligators
(one from Lake Gnffin [G] and one from Rockefeller [R]) BSA was included as a
negative control Brackets surround expected molecular weight (MW) range for Vtg
proteins (A) Blot was incubated in phospho-senne pnmary antibody (B) Blot was
incubated in* *phospho-tyrosine pnmary antibody (C) Blot was incubated in
a-phospho-threonne pnmary antibody (D) Blot was incubated without primary
antibody a-phospho-senne primary antibody reacted the strongest with all three proteins
in the 250-450 kDa MW range while the a-phospho-tyrosine pnmary antibody only
reacted with the 250 kDa protein and the a-phospho-threonne pnmary antibody did not
react with any of the three proteins















CHAPTER 4
CONCLUSIONS AND FUTURE DIRECTIONS

This is the first study that has attempted to identify Vtg in adult female American

alligators through the utilization of sequence analysis coupled with limited biochemical

characterization. It is therefore essential that additional sequencing be completed. These

data will then be useful in developing a sensitive and specific quantitative assay for

alligator Vtg. Such an assay could then be utilized throughout the entire reproductive

cycle for several sites to establish a seasonal monitoring protocol. This could be

expanded to a time course study designed to follow the production of Vtg and its

subsequent modifications and eventual deposition in the growing follicles.

There are several possible explanations which would support that the three female

specific proteins analyzed in this study are or are not Vtg or Vtg metabolites. Based on

the information gathered the most likely explanation is that they are Vtg metabolites

(sequencing data confirms this to be true for the 250 kDa protein) that are at different

stages of post-translational modification. It is likely that their inevitable fate will be

deposition in the growing oocyte to be used by the embryo as a nutritional source.

However, another possibility is that one or more of them are polypeptides which have

been cleaved into one or more products upon analysis by denaturing SDS-Page.

The phospholipase digestion analysis provided data that is in direct conflict with

what has been previously described in the literature. As already discussed in Chapter 3,

there are plausible explanations as to why this assay may have yielded negative results;

complexity of the sample or lack of sensitivity of the assay. However there is another









possibility; perhaps Vtg is constructed differently than we have all assumed. It is

published many times over that Vtg is a phospholipoprotein. This implies that there are

phosphorylated lipid moieties attached to the protein backbone. If this were true, and the

negative results were not due to interference, then the phospholipase would have cleaved

these moieties from the backbone leaving a smaller phospholipid product and the

remainder of the protein construct as a second product. Perhaps Vtg is more complex

than was previously assumed. The western blot analysis identifying phosphorylated

amino acids confirms that there are phosphate groups attached directly to the protein

backbone. Further investigation of the structure and subsequent folding of the protein is

warranted. This type of research would help to elucidate potential structure activity

relationships between Vtg and other proteins as well as EDCs such as OCPs.

During the initial analysis of plasma protein profiles there were some subtle

differences in lower MW proteins which did not fall within the target MW range for this

study thereby suggesting qualitative differences in the post-translational processing of

other female specific proteins in animals from Lake Griffin compared to Rockefeller.

These results warrant further investigations of these plasma protein profiles from female

animals from these sites as well as others to determine whether the differences may be

contaminant related or whether they are just an artifact of regional genetic variations.

Once this question is answered, it would be beneficial to examine the livers from the

same animals looking specifically at Vtg precursors and other reproductive proteins

(including metabolic enzymes) to begin to elucidate a potential mechanisms) for

metabolic alterations which may affect reproductive success in animals from OPC

contaminated sites. Therefore future directions should include the same types of analysis






64


on the liver and ovidutcal tissues to further enhance knowledge of the alligator

reproductive system at the molecular level. To date this is an underdeveloped area which

could help to elucidate the mechanisms) behind altered reproductive success in these

animals. Eventually there needs to be a binding assay developed in alligators which

would be able to explore the interactions of Vtg and various tissues and subsequent

involvement with other proteins such as potential carrier or chaperone proteins. This

could be expanded to explore possible interaction of these proteins with OCPs and other

EDCs.
















REFERENCE LIST


Allner B, Wegener G, Knacker T, Stahlschmidt-Allner P (1999). Electrophoretic
determination of estrogen-induced protein in fish exposed to synthetic and naturally
occurring chemicals. Sci Total Environ. 233, 21-31.

Baerga-Santini C and Hemandez de Morales M (1991). Vitellogenin diversity in tropical
lizards (Anolis pulchellus): identification and partial characterization. Comp Biochem
Physzol B BochemMol Bol. 100, 347-359.

Benton J, Douglas D. (1994) Ocklawaha fisheries investigations completion report, 1991-
1994: Study XIII, Assessment of fisheries restoration potential for reclaimed agricultural
lands in the upper Ocklawaha Basin; Study XIV, Ocklawaha chain of lakes largemouth
bass population studies; Study XV, Black crappie production in Lake Griffin. State of
Florida Game and Fresh Water Fish Commission.

Bowman CJ, Kroll KJ, Gross TG, Denslow ND (2002). Estradiol-induced gene
expression in largemouth bass (Micropterus salmoides). Mol Cell Endocrinol. 196, 67-
77.

Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal Blochem. 7, 248-
254.

Bradsfield SM, Weber LP, Talent LG, Janz DM (2002). Dose-response and time course
relationships for vitellogenin induction in male western fence lizards (Sceloporus
occidentalis) exposed to ethinylestradiol. Environ Toxicol Chem. 21, 1410-1416.

Brion F, Rogerieux F, Noury P, Migeon B, Flammarion P, Thybaud E, Porcher JM,
(2000). Two-step purification method of vitellogenin from three teleost fish species:
rainbow trout (Oncorhynchus mykiss), Gudgeon (Gobio gobio) and chub (Leuciscus
cephalus). J Chromotogr B Biomed Sci Appl. 737, 3-12.

Brown MA, Came A, Chambers GK (1997). Purification, partial characterization and
peptide sequences of vitellogenin from a reptile, the tuatura (Sphenodon punctatus).
Comp Biochem Physiol B Biochem Mol Biol. 117, 159-168.

Buhi WC, Alvarez IM, Binelli M, Walworth ES, Guillette LJ Jr. (1999). Identification
and characterization of proteins synthesized de novo and secreted by the reproductive
tract of the American alligator, Alligator mississippiensis. JReprodFertil. 115, 201-213.









Carevali O, Mosconi G, Angelini F, Limatola E, Ciarcia G, Polzonetti-Magni A (1991).
Plasma vitellogenin and 17 beta-estradiol levels during the annual reproductive cycle of
Podacis s. sicula Raf. Gen and Comp Endocrinol. 84, 337-343.

Cobb GP, Houlis PD, Bargar TA (2002). Polychlorinated biphenyl occurence in
American alligators (Alhgator mississippiensis) from Louisiana and South Carolina.
Environ Pollut. 118, 1-4.

Crain DA and Guillette LJ Jr. (1998). Reptiles as models of contaminant-induced
endocrine disruption. Anim Reprod Sci. 53, 77-86.

Crain DA, Guillette LJ Jr. Rooney AA Pickford DB (1997). Alterations in
steroidogenesis in alligators (Alligator mississippiensis) exposed naturally and
experimentally to environmental contaminants. Environ Health Perspect. 105, 528-533.

Dalrymple GH (1996). Growth of American alligators in the Shark Valley region of
Everglades National Park. Copeza. 212-216.

Davis LM, Glenn TC, Elsey RM, Dessauers HC, Sawyer RH (2001). Multiple paternity
and mating patterns in the American alligator, Alhgator mississippiensis. Mol Ecol. 10,
1011-1024.

Densmore LD. (1981). Biochemical and immunologicalsystematics of the order
crocodilia. New Orleans, Louisiana State University.

Duggan A, Paolucci M, Tercyak A, Gigliotti M, Small D, Callard I (2001). Seasonal
variations in plasma lipids, lipoproteins, apolipoprotein A-I and Vitellogenin in the
freshwater turtle, Chrysemys picta. Comp Biochem PhysiolA Mol Integr Physiol. 130,
253-269.

Eckel RH (1989). Lipoprotein lipase. A multifunctional enzyme relevant to common
metabolic diseases. NEnglJMed. 320, 1060-1068.

Elsey RM, Lance VA, Campbell L (1999). Mercury levels in alligator meat in South
Louisiana. Bull Environ Contam Toxicol. 63, 598-603.

Ertl RP, Bandiera SM, Buhler DR, Stegman JJ, Winston GW (1999). Immunochemical
analysis of liver microsomal cytochromes P450 of the American alligator, Alhlgator
mississippiensis. ToxicolApplPharmacol. 157, 157-165.

Ferguson MWJ (1985). Reproductive Biology and Embryology of the Crocodilians. In
'Biology of the Reptilia'. pp. 329-491. (John Wiley & Sons: New York.)

Ferguson MWJ and Joanen T (1982). Temperature of egg incubation determines sex in
Alhgator mississippiensis. Nature London 296, 850-853.

Ferguson MWJ and Joanen T (1983). Temperature dependent sex determination in
Alhgator mississippiensis. J Zool. 200, 143-177.









Gagne F and Blaise C (1998). Estrogenic properties of municipal and industrial
wastewaters evaluated with a rapid and sensitive chemoluminescent in situ hybridization
assay (CISH) in rainbow trout hepatocytes. Aquat Toxicol. 44, 83-92.

Gagne F and Blaise C (2000). Evaluation of environmental estrogens with a fish cell line.
Bull Environ Contam Toxicol. 65, 494-500.

Gagne F, Marcogliese DJ, Blaise C, Gendron AD (2001). Occurrence of compounds
estrogenic to freshwater mussels in surface waters in an urban area. Environ Toxicol. 16,
260-268.

Garrick LD and Lang JW (1977). Social signals and behaviors of adult alligators and
crocodiles. Am Zool. 17, 225-239.

Giroux DJ. (1998) Lake Apopka revisited: a correlational analysis of nesting anomalies
and DDT contaminants. Gainesville, Florida, University of Florida.

Glasgow LR, Paulson JC, Hill RL (1977). Systematic purification of five glycosidases
from Streptococcus (Diplococcus) pneumonae. JBiol Chem. 252, 8615-8623.

Gronen S, Denslow N, Manning S, Barnes S, Barnes D, Brouwer M (1999). Serum
vitellogenin levels and reproductive impairment of male Japanes medaka (Oryzias
latipes) exposed to 4-tert-octylphenol. Environ Health Perspect. 107, 385-390.

Groombridge B (1987). The distribution and status of world crocodilians. In 'Wildlife
Management: Crocodiles and Alligators'. pp. 9-21. (Surrey Beatty and Sons: Australia.)

Gross TS, Guillette LJ Jr., Percival HF, Masson GR, Matter JM, Woodward AR (1994).
Contaminant-induced reproductive anomalies in Florida. Comp Pathol Bull. 26, 2-8.

Guillette LJ Jr., Gross TS, Gross DA, Rooney AA, Percival HF (1995). Gonadal
steroidogenesis in vitro from juvenile alligators obtained from contaminated or control
lakes. Environ Health Perspect, Suppl. 103, 31-36.

Guillette LJ Jr., Gross TS, Masson GR, Matter JM, Percival HF, Woodward AR (1994).
Developmental abnormalities of the gonad and abnormal sex hormone concentrations in
juvenile alligators from contaminated and control lakes in Florida. Environ Health
Perspect. 102, 680-688.

Guillette LJ Jr. and Gunderson MP (2001). Alterations in development of reproductive
and endocrine systems of wildlife populations exposed to endocrine-disrupting
contaminants. Reproduction 122, 857-864.

Guillette LJ Jr., Vonier PM, McLachlan JA (2002). Affinity of the alligator estrogen
receptor for serum pesticide contaminants. Toxicology 181-182, 151-154.









Guillette LJ Jr., Woodward AR, Crain DA, Masson GR, Palmer BD, Cox C, You-Xiang
Q (1997). The reproductive cycle of the American alligator (Alhgator mississippiensis).
Gen Comp Endocrinol. 108, 87-101.

Guillette LJ Jr. and Milnes MR (2001). Recent observations on the reproductive
physiology and toxicology of crocodilians. In 'Crocodilian Biology and Evolution'. pp.
199-213. (Surrey Beatty and Sons: Australia.)

Hall RJ and Henry PFP (1992). Assassing effects of pesticides on amphibians and
reptiles: status and needs. Herpetol J. UK, 65-71.

Hartling RC, Pereira JJ, Kunkel JG (1997). Characterization of a heat-stable fraction of
lipovitellin and development of an immunoassay for vitellogenin and yolk protein in
winter flounder (Pleuronectes americanus). J Exp Zool. 278, 156-166.

Heck J, MacKenzie DS, Rostal D, Medler K (1997). Estrogen induction of plasma
vitellogenin in the Kemp's Ridley sea turtle (Lepidochelys kempi). Gen Comp Endocrinol.
107, 280-288.

Heinz GH, Percival HF, Jennings ML (1991). Contaminants in American alligator eggs
from Lake Apopka, Lake Griffin, and Lake Okeechobee, Florida. EnvironMonit and
Assess. 16, 277-285.

Heppel SA, Denslow ND, Folmar LC, Sullivan CV (1995). Universal assay of
vitellogenin as a biomarker for environmental estrogens. Environ Health Perspect. 103,
9-15.

Herbst LH, Siconolfi-Baez L, Torelli JH, Klein PA, Kerben MJ, Schumacher IM (2003).
Induction of vitellogenesis by estradiol-171 and development of enzyme-linked
immunosorbant assays to quantify plasma vitellogenin levels in green turtles (Chelonma
mydas). Comp Blochem Physiol B Blochem Mol Biol. 135, 551-563.

Hutton JM (1986). Age determination of living Nile crocodiles from the cortical
stratification of bone. Copeia 2, 332-341.

Hutton JM (1987). Techniques for ageing wild crocodilians. In 'Wildlife Management:
Crocodiles and Alligators'. pp. 211-216. (Surrey Beatty and Sons Pty Limited: Australia.)

Irwin LK, Gray S, Oberd6rster E (2001). Vitellogenin induction in the painted turtle,
Chrysemys picta, as a biomarker of exposure to environmental levels of estradiol. Aquat
Toxicol. 55, 49-60.

Iwase H and Hotta K (1993). Release of o-linked glycoprotein glycans by endo-alpha-N-
acetylgalactosaminidase. Methods Mol Biol. 14, 151-159.

James AM and Oliver JH Jr. (1997). Purification and partial characterization ofvitellin
from the black-legged tick, Ixodes scapularis. InsectBiochem Mol Biol. 27, 639-649.









Joanen T and McNease L (1989). Ecology and physiology of nesting and early
development of the American alligator. Am Zool. 29, 989-998.

Joanen T and McNease LL (1980). Reproductive biology of the American alligator in
south-west Louisiana. In 'Reproductive Biology and Diseases of Captive Reptiles'. (JB
Murphy and JT Collins, Eds.), 153-159.

Kawahara A, Sato K, Amano M (1983). Regulation of protein synthesis by estradiol 17
beta, dexamethasone and insulin in primary cultured Xenopus hepatocytes. Exp Cell Res.
148, 423-436.

Kernaghan NJ, Monck E, Weiser C, Gross TS (2002). Characterization and manipulation
of sex steroids and vitellogenin in freshwater mussels. Society of Environmental
Toxixology and Chemistry 23rd Annual Meeting in North America, Salt Lake City, Utah.
November 16-20, 2002, 263.

Kushlan JA and Jacobson T (1990). Environmental variability and the reproductive
success of everglades alligators. JHerpetol. 24, 176-184.

Laemmeli UK (1970). Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227, 680-685.

Lance VA (1989). Reproductive cycle of the American alligator. Am Zool. 29, 999-1018.

Lance VA (2003). Alligator physiology and life history: the importance of temperature.
Exp Gerontol. 38, 801-805.

Lance VA, Joanen T, McNease L (1983). Selenium, vitamin E, and trace elements in the
plasma of wild and farm reared alligators during the reproductive cycle. Can J Zool. 61,
1744-1751.

Lance VA and Lauren D (1984). Circadian variation in plasma corticosterone in the
American alligator, Alligator mississippiensis and the effects of ACTH injections. Gen
Comp Endocrinol. 54, 1-7.

LeClerq F, Schnek AG, Braunitzer G, Stangl A, Schrank B (1981). Direct reciprocal
allosteric interaction of oxygen and hydrogen carbonate sequence of the haemoglobins of
caiman (Caiman crocodylus), the Nile crocodile (Crocodylus niloticus) and the
Mississippi crocodile (Alligator mississippiensis). ZPhyszol Chem. 1151-1158.

Le Guellec K, Lawless K, Valotaire Y, Kress M, Tenniswood M (1988). Vitellogenin
gene expression in male rainbow trout (Salmo gairdneri). Gen Comp Endocrinol. 71,
359-371.

Lim EH, Lam TJ, Ding JL (1997). Direct submission accession # T31095. NCBI
database.









Marburger JE, Johnson WE, Gross TS, Douglas DR, Di J (2002). Residual
organochlorine pesticides in soils and fish from wetland restoration areas in Central
Florida, USA. Wetlands. 22, 705-711.

Masson GR. (1995). "Environmental influences on reproductive potential, clutch viability
and embryonic mortality of the American alligator in Florida". Gainesville, Florida,
University of Florida.

Matter JM, McMurry CS, Anthony AB, Dickerson RL (1998). Development and
implementation of endocrine biomarkers of exposure and effects in American alligators
(Alligator mississippiensis). Chemosphere. 37, 1905-1914.

Morales MH, Baerga-Santini C, Cordero-Lopez N (1996). Synthesis ofvitellogenin
polypeptides and deposit of yolk proteins in Anolis pulchellus. Comp Blochem Physiol B
BlochemMolBiol. 114, 225-231.

Morales MH, Pagan SM, G6mez Y (2002). Immunodissection of yolk lipovitellin (LVi)
demonstrates the existence of different LV1-domains and suggests a complex family of
vitellogenin genes in the lizard Anols pulchellus. Comp Blochem Physiol B Blochem Mol
Biol. 131, 339-348.

Morales MH and Sanchez EJ (1996). Changes in vitellogenin expression during
captivity-induced stress in a tropical anole. Gen Comp Endocrnol. 103, 209-219.

Mouchel N, Trichet V, Betz A, Le Pennec JP, Wolff J (1996). Characterization of
vitellogenin from rainbow trout (Oncorhynchus mykiss). Gene. 174, 59-64.

Murakami H and Nakai M (2001). Direct submission accession # BAB79591. NCBI
database.

Nilsson NO, Stralfors P, Fredrikson G, Belfrage P (1980). Regulation of adipose tissue
lipolysis: effects of noradrenaline and insulin on phosphorylation of hormone-sensitive
lipase and onlipolysis in intact rat adipocytes. FEBSLett. 111, 125-130.

Olivecrona T, Bengtsson-Olivecrona G, Ostergaard P, Lui G, Chevreuil O, Hultin M
(1993). New aspects on heparin and lipoprotein metabolism. Haemostasi.s. 23, 150-160.

Palmer B and Guillette LJ Jr. (1992). Alligators provide evidence for the evolution of an
archosaurian mode of oviparity. Biol Reprod. 46, 39-47.

Palmer BD and Palmer SK (1995). Vitellogenin induction by xenobiotic estrogens in the
red-eared turtle and African clawed frog. Environ Health Perspect. 103, 19-25.

Peabody FE (1961). Annual growth zones in living and fossil vertebrates. JMorphology.
108, 11-62.









Perutz MF, Bauer C, Gros G, LeClerq F, Vandecassarie C, Schnek AG, Brainitzer G,
Friday AE, Josey KA (1981). Allosteric regulation of crocodilian haemoglobin. Nature
London. 291, 682-684.

Pruneta V, Autran D, Ponsin G, Marcais C, Duvillard L, Verges B, Berthezene F, Moulin
P (2001). Ex vivo measurement of lipoprotein lipase-dependent very low density
lipoprotein (VLDL)-triglyceride hydrolysis in human VLDL: An alternative to the
postheparin assay of lipoprotein lipase activity? J Chn Endocrinol Metab. 86, 797-803.

Rauschenberger RH, Wiebe JJ, Buckland JE, Smith JT, Sepulveda MS, Gross TS. (2003)
Achieving environmentally relevant organochlorine pesticide concentrations in eggs
through maternal exposure in Alhgator mississippiensis. Mar Environ Res. (In Press).

Romano M, Rosanova P, Anteo C, Limatola E (2002). Lipovitellins and phosvitins of the
fertilized eggs during embryo growth in the oviporous lizard Podarcis sicula. Mol
ReprodDev. 63, 341-348.

Rosanova P, Romano M, Marciano R, Anteo C, Limatola E (2002). Vitellogenin
precursors in the liver of the oviporous lizard, Podarcis sicula. Mol ReprodDev. 63, 349-
354.

Roubal WT, Lomax DP, Willis ML, Johnson LL (1997). Purification and partial
characterization of English sole (Pleuronectes vetulus) vitellogenin. Comp Blochem
Physiol B BochemMol Bol. 118, 613-622.

Ryffel GU (1978). Synthesis of vitellogenin, an attractive model for investigating
hormone0induced gene activation. Mol Cell Endocrnol. 12, 237-246.

Sepfilveda M, Johnson WE, Higman JC, Denslow ND, Schoeb TR, Gross TS (2002). An
evaluation of biomarkers of reproductive function and potential contaminant effects in
Florida largemouth bass (Micropterus salmoidesfloridanus ) sampled from the St. Johns
River. Sci Total Environ. 289, 133-144.

Sierra-Santoyo A, Hernndez M, Albores A, Cebrian ME (2000). Sex-dependent
regulation of hepatic cytochrome P-450 by DDT. Toxicol Sci. 54, 81-87.

Sumpter JP and Jobling S (1995). Vitellogenesis as a biomarker for estrogenic
contamination of the aquatic environment. Environ Health Perspect. 103, 173-178.

Szkudinski MW, Thotakura NR, Tropea JE, Grossman M, Weintraub BD (1995).
Asparagine-linked oligosaccharide structures determine clearance and organ distribution
of pituitary and recombinant thyrotropin. Endocrinology. 136, 3325-3330.

Talent LG, Dumont JN, Bantle JA, Janz DM, Talent SG (2002). Evaluation of western
fence lizards (Sceloporus occidentalis) and eastern fence lizards (Sceloporus undulatus)
as laboratory reptile models for toxicological investigations. Environ Toxicol Chem. 21,
899-905.









Tarentino AL and Plummer TH Jr. (1994). Enzymatic deglycosylation of asparagine-
linked glycans: purification, properties, and specificity of oligosaccharide-cleaving
enzymes from Flavobacterium meningosepticum. Methods Enzymol. 230, 44-57.

Uchida Y, Tsukada Y, Sugimori T (1979). Enzymatic properties of neurominidases from
Arthrobacter ureafaciens. JBlochem (Tokyo). 86, 1573-1585.

Uribe MCA and Guillette LJ Jr. (2000). OOgenesis and ovarian histology of the
American alligator Alhgator mississippiensis. JMorphol. 245, 225-240.

Vliet K (1989). Social displays of the American alligator (Alligator mississipplensis). Am
Zool. 29, 1019-1031.

Wahli W, Dawid IB, Ryffel GU, Weber R (1981). Vitellogenesis and the vitellogenin
gene family. Science 212, 298-304.

Wahli W, Martinez E, Corthesy B, Cardinaux JR (1989). Cis- and trans-acting elements
of the estrogen-regulated vitellogenin gene B1 of Xenopus laevis. JSteroidBlochem. 34,
17-32.

Walker P, Brown-Luedi M, Germond JE, Wahli W, Meijlink FC, van het Schip AD,
Roelink H, Gruber M, Ab G (1983). Sequence homologies within the 5' end region of the
estrogen-controlled vitellogenin gene in Xenopus and chicken. EMBO J. 2, 2271-2279.

Walker P, Germond JE, Brown-Luedi M, Givel F, Wahli W (1984). Sequence
homologies in the region preceding the transcription initiation site of the liver estrogen-
responsive vitellogenin and apo-VLDLII genes. Nucleic Acids Res. 12, 8611-8626.

Wang H, Yan T, Tan JT, Gong Z (2000). A zebrafish vitellogenin gene (vg3) encodes a
novel vitellogenin without a phosvitin domain and may represent a primitive vertebrate
vitellogenin gene. Gene. 256, 303-310.

Wang SY and Williams DL (1982). Biosynthesis of the vitellogenins. Identification and
characterization of nonphosphorylated precursors to avian vitell9ogenin I and
vitellogeninII. JBiol Chem. 257, 3837-3846.

Wink C and Elsey RM (1986). Changes in femoral bone morphology during egg-lying in
Alhgator mississippiensis. JMorphol. 189, 183-188.

Wood JM, Woodward AR, Humphrey SR, Hines TC (1985). Night counts as an index of
American alligator population trends. Wildl Soc Bull. 13, 262-272.

Woodruff AR and Moore CT. (1989) "Statewide alligator surveys". Final report.
Tallahassee, Florida, Florida Game and Freshwater Fish Commission.

Woodward AR, Jennings ML, Percival HF, Moore CT (1993). Low clutch viability of
American alligators on Lake Apopka. Fl Sci. 56, 52-63.






73


Woodward AR, Moore CT, Delany MF (1992). "Experimental alligator harvest". Final
Report. Florida Game and Fresh Water Fish Commission.

Yamamura J, Adachi T, Aoki N, Nakajima H, Nakamura R, Matsuda T (1995).
Precursor-product relationship between chicken vitellogenin and the yolk protein: the 40
kDa yolk plasma glycoprotein is derived from the C-terminal cystein-rich domain of
vitellogenin II. Blochim Blophys Acta 1244, 384-394.

Yoon S (2002). Direct submission accession # BAC20186. NCBI database.

Yoshitome S, Nakamura H, Nakajo N, Okamoto K, Sugimoto I, Kohara H, Kitayama K,
Igarashi K, Ito S, Sagata N, Hashimoto E (2003). Mr 25,000 protein, a substrate for
protein serine/threonine kinases, is identified as a part of Xenopus laevis vitellogenin Bl.
Dev Growth Differ. 45, 283-294.
















BIOGRAPHICAL SKETCH

Eileen K. Monck was born June 6, 1962 in Bronx, New York. After graduating

from New Britain High School in 1980, she explored different career possibilities before

enrolling at Central Connecticut State University. She graduated in 1989, with a

Bachelor of Science degree in biology and secondary education (with minors in

chemistry and general science).

In 1989 she began working at the University of Florida as a research technician,

where she developed a desire to further her education. In 1999 she began her graduate

work in environmental toxicology, which she expanded to reproductive endocrinology of

the American alligator. She will graduate in December 2003 with a Master of Science

degree. Eileen will continue her work with alligators, under the continued supervision of

Dr. Timothy Gross at the United States Geological Survey in Gainesville, Florida.

Through her college career, Eileen has been a wife, and a mother to three

children; and has enjoyed exposing her children to all of the fascinating educational

opportunities her career has to offer. She has often been involved in bringing science into

classrooms at many age levels, and looks forward to many more opportunities to do so.