Soy Phytoestrogens in Reproduction

Progress made in meeting original objectives:

	We have made good progress in meeting specific research objectives 
of the project.   Experiments under specific aims 2, 3, and 5 are underway, 
and experiments described under specific aim 1 and 4 will be started by the 
end of the summer.  
	Specific Aim 1.  We have not actually started the HPLC experiments 
described under  this specific aim.  The delay is due to modifications in 
the experimental design because of budget cuts in the first year.  Bruce 
Jacobson, with Dr. Lightfoot's assistance, will be starting those 
experiments in the next few months.  
	Specific Aim 2.  Dr. Lightfoot's laboratory has been successful in 
the QTL mapping of three enzymes in the biosynthetic pathway of soy 
flavanoids.  These include phenylalanine ammonia lyase (PAL), chalcone 
synthase (CHS), and chalcone isomerase (CHI).    Five genes and 
2 polymorphisms have been linked to PAL; 5 to 7 genes and 3 polymorphisms 
have been linked to CHS; and 1 gene which is polymorphic has been linked to 
CHI.  The product of CHI is the flavone naringenin.  Naringenin is an 
isomer of the isoflavone genistein, the most abundant isoflavone in 
soybeans.  
	Specific Aim 3.  We have been successful in growing porcine 
mesonephric (reproductive progenitor) cells in culture from fetuses, 
30 to 45 days gestational age.   Dr. Winters and his graduate students 
have spent a significant amount of time developing the fetal porcine cell 
culture system in the lab.   We have had some problems and setbacks with 
microorganism contamination of the primary cultures due to what we believe 
is location of the tissue culture lab--a room with large, poorly sealed 
windows to the outside, and close to student traffic.  We have recently 
moved that laboratory to a more ideal location.  Mark Williams (graduate 
student supported 9 mo by departmental assistantship) has been using 
thymidine incorporation assays to measure DNA synthesis in the fetal 
reproductive progenitor cell culture system.  He has preliminary results 
that show that soy phytoestrogens genistein and daidzein modulate DNA 
synthesis in primary mesonephric cells derived from 60 day gestational age 
fetuses.  Mark is also examining cellular and molecular effects of these 
soy phytoestrogens on the gilt uterus.   Natasha Danilovich, (graduate 
student supported by another C-FAR project),  is in the early stages of 
developing an in situ apoptosis assay in the ovary.  We hope to use this 
same assay to study programmed cell death in these fetal progenitor cells.
	Specific Aim 4.  Experiments under this aim will be starting soon 
now that tissue culture is successful.  
	Specific Aim 5.  Jennifer Greer (unsupported graduate student) and 
Dr. Banz are just getting the rat feeding studies described under this aim 
underway.  Jennifer and Shunfang Hou (graduate student supported by a 
related ORDA grant) are developing the Reverse Transcriptase-Polymerase 
Chain Reaction (RT-PCR) generated cDNA probes to the porcine and rat 
estrogen receptor to use as a molecular markers to study the physiological 
endpoints under this objective.  We are confident that we will be successful
in generating these specific cDNA probe in the next month or so.