Soy Phytoestrogens: Analysis in Soybeans and Effects on Swine Reproduction

Introduction

	Soy products contain estrogenic isoflavones called phytoestrogens 
that have been shown to have physiological effects on humans and animals 
that ingest these products.  Soy phytoestrogens may have beneficial effects 
in humans by reducing susceptibility to certain cancers(1), reducing serum 
cholesterol(2), and ameliorating symptoms of menopause(3).  It is evident 
that phytoestrogens also affect reproduction in farm animals(4,5).  These 
studies have concentrated on the effects of phytoestrogens from forage 
legumes on ruminants (cows and sheep).  Pigs (humans and rats) are 
monogastric, and are more likely to be affected by phytoestrogens because 
these isoflavones would be less likely broken down in the gastrointestinal 
tract and therefore, more likely absorbed into the blood stream(6).  
In ruminants, these phytoestrogen would be subjected to the bacterial 
degradation of the rumen.  Soybean-derived feedstuffs make up a significant 
percentage ( 20%) of most swine diets.  Of the 54.5 million hogs and pigs 
in the U.S., 5.7 million are produced in Illinois(7).  Illinois also ranks 
number one in soybean production in the U.S.(8).  Therefore, our  research 
examining the effects of a soybean product on swine reproduction directly 
affects agriculture in the state of Illinois.  Certain soy phytoestrogens 
may be more effective than others, and some may even have adverse 
consequences(4).  If soy products are to attain their full potential, we 
must develop a greater understanding of specific attributes of individual 
soy phytoestrogens.  Our research is concentrating on the effects of these 
compounds on reproduction, and provide the knowledge base necessary to 
manipulate phytoestrogen concentrations in soybeans.  This research has 
great potential to expand the market for soy products, especially within 
the growing phytopharmaceutical industry.

Objectives

	Our overall objectives are to chemically and genetically 
characterize phytoestrogen production in different varieties of soybeans 
and to determine the effects of soy-related phytoestrogens on mammalian 
reproductive function.  Specific aims are:

	1.  Isolation of Soy-related Phytoestrogens
		To determine high performance liquid chromatography (HPLC) 
		elution profiles of eight soy-related phytoestrogens in 
		soybean seed derived from eleven varieties of soybeans and 
		their crosses.
	2.  Gene Mapping 
 		To identify molecular markers in the existing maps of the 
		soybean genome that would be linked to genes controlling 
		phytoestrogen profiles (PEPs) generated from the eleven 
		soybean varieties and their crosses.
	3.  In Vitro Testing of Phytoestrogens on Reproductive Differentiation
		To determine the kinetics of specific phytoestrogen binding 
		to an estrogen receptor and the effects soy-related 
		phytoestrogens have on cell proliferation and apoptosis 
		(programmed cell death) in cultured primary cells derived 
		from fetal porcine reproductive progenitor organs.
	4.  In Vitro Testing of Phytoestrogens on Ovarian Function
		To determine the effects of phytoestrogens on 
		steroidogenesis (ovarian health) and apoptosis 
		(ovulation propensity) in cultured primary granulosa cells 
		from porcine ovaries. 	
	5.  In Vivo Testing of Phytoestrogens on Reproduction
		To determine the effects of phytoestrogens on the 
		reproduction of animals fed these soy products.

Achieving these objectives will be economically beneficial to swine and 
soybean producers by making pork production more efficient and by increasing
the utilization and/or demand of soy products in the swine industry.

Genetic and Chemical Analysis of Phytoestrogens in Soybeans

	In the past decade, much effort has been put into the genetic 
analysis of soybeans, primarily for the detection of genes involved in 
disease resistance and susceptibility(9-12).  Gene maps have been very 
useful for plant genome analysis and for the detection of quantitative 
trait loci (QTL) as tools in plant breeding for disease resistance(9-12) 
and altered soybean composition(13).  Methods have also been developed to 
extract, separate and quantify phytoestrogens from soybeans and their 
products(14,15).  A primary purpose of our research is to identify markers 
genetically linked to loci conditioning variation in phytoestrogen 
production by different soybean varieties.  Preliminary results have 
identified polymorphisms (differences) between the Essex and Forrest 
varieties of soybeans in three of the genes important in the biosynthesis 
of phytoestrogens (G. Torto, T. Winters, & D. Lightfoot, unpublished).  
Chemical analysis studies and further genetic studies are underway in our 
laboratories. This genetic and chemical information could then be used in 
plant breeding to control the levels of phytoestrogens produced by soybeans.

Phytoestrogens and Reproduction
  
	Estrogens have been shown to affect ovarian function and early 
embryonic development.  In swine, estradiol has been found to increase 
steroidogenesis in ovarian granulosa cells(16), and in rats, decrease 
apoptosis or programmed cell death of ovarian cells(17).  A decrease in 
apoptosis could increase ovulation rate.  Increased ovulation rate in 
swine (if coupled with increased rates of embryo survival) makes possible 
an increase in average litter size.  Estrogens also play an important role 
in early embryonic development in swine.  Specific examples of 
estrogen-regulated events occurring early in porcine pregnancy include 
intrauterine migration and spacing(18), morula-blastocyst transition(19), 
and maternal recognition of pregnancy(20). Estrogens also play a role in 
embryo implantation in rodents(21).  We hypothesize that phytoestrogens in 
soybean meal can positively affect ovulation rate and embryonic survival 
in swine, thus increasing average litter size. The positive influence of 
phytoestrogens on these events would increase embryonic survival and thus 
litter size. An average increase of one pig per litter nationwide would 
allow us to produce 17 million more market pigs to meet expanding world 
needs (i.e., export market).  In addition, a positive influence of soy 
phytoestrogens on reproduction will increase demand to isolate these 
compounds. 
	Evidence also suggests that phytoestrogens affect the female 
reproductive tract, a primary target of estrogens(5,22).  The role of 
estrogen in the proper development of the reproductive tract has long been 
suspected because of known permanent reproductive alterations, including 
cancer, caused by fetal exposure to the synthetic estrogen, 
diethylstilbestrol (DES)(23,24).  Most notable is a high incidence of 
vaginal and cervical cancer in daughters of women exposed to DES during 
pregnancy (25).  Evidence also indicates that males exposed to DES in 
utero have decreased fertility, and other reproductive defects(23).  These 
defects suggest that tissues derived from the reproductive system 
progenitors were affected.   In Australia, phytoestrogens found in red 
clover have been reported to cause permanent deleterious reproductive 
tract alterations in sheep(4).  However, these clover compounds 
(coumestrans) are from a different biochemical family than the 
phytoestrogens (isoflavones) found in soybeans.
	Soy-produced phytoestrogens appear to be protective against 
endocrine-related cancers (e.g. breast & prostate cancer)(1).  It is 
hypothesized that these weak estrogens elicit anticancer effects by binding 
to estrogen receptors, thus blocking the proliferative effects of stronger 
estrogen like estradiol (naturally occurring estrogen) and DES.  It is 
possible that phytoestrogens may have the same effect in fetal sexual 
differentiation, protecting the developing reproductive system from strong 
estrogens that may have deleterious effects on proper differentiation.  
However, there may also be detrimental effects of these phytoestrogenic 
compounds on sexual differentiation and reproduction(4).  In either case, 
the information generated from our research would be useful in the breeding 
of soybean varieties which have been characterized for phytoestrogen 
production.
	Only one study has reported possible effects of soy phytoestrogens 
on reproduction in swine(5).  This study examined the effects of soybean 
meal on the reproductive development of prepubertal gilts.  Although, 
estrogenic effects were observed (increased vulva diameters), results were 
inconclusive.  Therefore a more comprehensive study of the effects of soy 
phytoestrogens on swine reproduction is warranted.  Preliminary results 
from our laboratory using swine tissues suggest that genistein 
(the most abundant phytoestrogen in soybeans) affects both ovarian 
apoptosis (A. Suttner, N. Danilovich, & T. Winters, unpublished) and cell 
division of the fetal male reproductive system (M. Williams, & T. Winters, 
unpublished).  The significance of this preliminary research is unclear, 
but may have implications in ovulation rate and the proper differentiation 
of the male reproductive system.

Summary

	In summary, soy phytoestrogens are known to affect the physiology 
of animals that ingest these compounds.  We are examining soy phytoestrogen 
and their reproductive effects at the genetic, biochemical, and 
physiological levels.  From these experiments, we hope to identify the 
beneficial and/or detrimental effects that soy phytoestrogens may have on 
reproduction.  Effects on parameters such as litter size may have a great 
economic significance in swine.  Once these physiological effects are 
sorted out, soybean varieties can be bred and/or genetically engineered to 
produce beneficial phytoestrogens. This would increase the utilization of 
soybeans in the livestock industry and the growing phytopharmaceutical 
industry; and therefore, would be of great economical importance to 
Illinois Agriculture.

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