Environmental estrogens 2012

The Israel Journal of Veterinary Medicine has issued a special issue in honor of Prof. Natan  Ayalon

here


This is the abstract of our contribution.

Effects of Environmental Estrogens on Reproductive Parameters in Domestic Animals

Shemesh, M. and Shore, L.S. Department of Hormone Research, Kimron Veterinary 
Institute, Bet Dagan, POB 12, 50250 Israel.

ABSTRACT

Environmental estrogens are natural products of plant (phytoestrogens) or animal origin (steroidal  estrogens) that have estrogenic properties. They are a major group in a category known as endocrine disruptors. In the field of animal husbandry, the effects of these environmental estrogens are well documented. This paper discusses  the effects of plant estrogens and environmental steroid hormones, such as estrogen and testosterone, as seen in animals in Israel. The areas considered are reproductive disorders, premature udder development, prolapsed oviduct, scrotal atrophy and skewed sex ratios.

Keywords: phytoestrogens, zearalenone, estradiol 17-β, testosterone, testes, oviductal prolapse, scrotum, sex ratio

Israel Journal of Veterinary Medicine  Vol. 67 (1):6-10 March 2012

Effect of estrogen implants on scrotal circumference and tonus in bull calves. Thirteen calves were implanted at 3 mo and their testicles measured at 6 months. Fourteen other calves similarly fed and housed served as controls.  The control circumference (cm) was significantly larger than the treated (P<0.01) and the tonus was significantly lower in treated bulls (P<0.02; 10=very poor; 20=poor; 30=good).

There is scant literature on the effects of exogenous estrogen on bull fertility.  Here is the graph from a short note  that I found.
Infertilité mâle bovine et teneur en coumestrol de la Luzerne.
Infertility in bulls and the concentration of coumestrol in alfalfa.

J.Le Bars, Nurard J.S.  Pharmacologie et Toxicolgie Vétérinaires, INRA Publ. Paris 1982 
Le Colloques de l'I.N.R.A. 8:157-158

 Belgian Blue or Belgian White Blue Bull. Established in the early 20th century, this heavily- (double) muscled, mottled Belgian breed, originally intended for beef and dairy use, is now mainly a beef breed, with bulls usually crossed as terminal sires to produce fast developing veal calves.

(cfgphoto.com copyright, used with permission)

For some reason, the picture of the Belgian Blue is getting a lot of hits.  Here are some more of  7 month old Belgian Blue calves selected for an insemination program taken in 1986 in Belgium (courtesy of Dr. S. Marcus)

Belgium blue calf 01

Belgium blue calf 02

Established in the early 21th century, this heavily-double muscled, mottled breed, originally intended for a scientific experiment. is now a  sporadic superhero.

Cleaning out the drawers and found this little note.

 Pregnancy in a hermaphroditic cow.  High estrogen content of the hermaphroditic testes .

Perl, Smuel; Marcus, Smuel; Shore, Laurence S.; Brenner, Gideon;  Shemesh, Mordechai

A pregnant heifer was examined after it was observed to have masses in the pelvic region.  At slaughter, it was found to have a gravid uterus with a 3 month old fetus.  Testes were observed in the mesovarium .  One of the testes was well formed with an epididymis and seminal vesicular structure. Histological examination of the testis showed Sertoli cells, Leydig cells, germ cells and seminal  vesicles.  The second testis was small and contain only medullary cell mass.  Steroid analysis of the formed testes indicated high levels of estrogen and normal levels of testosterone compared to normal testes [Table 1].

There has been a least one previous report of a pregnant hermaphroditic cow. [Trächtigkeit bein einem Zwitter des Rindes (Hermaphroditaismus verus alternans). Fürst.   Deutsche Tierärztliche Wochenschrift  121 15. April.  59:121, 1952] .   Estrogen has long been known to be present in bull seminal fluid [Payne A, Kelch R, Musich S and Halpern M. Oestrogen content of semen and the effect of exogenous oestradiol-17β on the oestrogen and androgen concentration in semen and blood plasma of bulls. Journal of Reproduction and Fertility  50:17-21 (1977)] and aromatase can be present in all of the various cells of the bovine testes. [Carreau, S.; Hess R.A.  Oestrogens and spermatogenesis Phil. Trans. R. Soc. B 2010 365, 1517-1535.]

Tissue source	Testosterone	Estrogen
	ng/g  fresh wt	ng/g  fresh wt
Hermaphrodite
Interstitial	35.0	2.0
Leydig	8.2	0.3
Epididymal	33.1	0.4
Normal Calf (1 yr)
Interstitial (left)	42.0	<0.1
Interstitial (right)	60.5	<0.1
Epididymal	2.8	<0.1
Epididymal	4.0	<0.1

Estrogen binding receptor 

For many years  the Hormone Research Laboratory made their own cytosolic estrogen receptor.  It was used for the determination of phytoestrogens like coumestrol in alfalfa and zearalenone in corn products.  However, it became  too difficult to  obtain 6 dy old pregnant rabbits so we abandoned the assay.  Efforts to find a replacement have not been successful to date. The most promising system is XDS's Lumi-cell ER based on engineered cells linked to luminiferase.

Seems to work nicely for zearalenone, coumestrol, genistein and diadzein in the expected order.
The upper graph also shows alpha estradiol which usually isn't tested for.  Alpha estradiol is the principal estrogen found in cow blood but is not considered an active estrogen. 

I guess I should mention invitrogen which makes LanthaScreen™ TR-FRET Estrogen Receptor alpha and beta assays.  On paper this sounds impressive.  An assay using recombinant receptor linked to the  rare earth element terbium.  When the receptor is engaged, the terbium shifts its spectrum which can be detected with a spectrophotometer.   They do not offer any training or someplace where you can go to see it in action.  After I ordered it and went over the specs, I saw that it could not possibly work on the spectrophotometer I had even though it stated on their web site that it would work.  After a short email to the company, I was informed that indeed it would not work.  If somebody out there wants the kit, they are welcome to it.  Google states not to put your email on a web site because of spammers so just write me on Facebook or something.  Laurence Shore

Media

http://seattletimes.nwsource.com/html/health/2018947430_spermbank19.html

Originally published August 18, 2012 at 4:41 PM | Page modified August 18, 2012 at 5:20 PM

Israel sperm banks find quality is plummeting
The quality of the product being offered to Israeli sperm banks is falling at an alarming rate, and no one is sure why.

By Edmund Sanders

Los Angeles Times

JERUSALEM — The founder of the Tel Aviv-based specialty firm raves about his product with the same gusto distillers reserve for their top-notch scotch. He's particularly proud of his "premium" line. Sure, it costs a bit more, but it's targeted at a more discriminating client.

Dr. Jacob Ronen is in the sperm business. Among other things, as head of Cryobank Israel, the country's largest private sperm bank, he guarantees that his stable of superior donors includes only tall, twenty-something ex-soldiers whose sperm has passed rigorous genetic testing.

But finding such super sperm isn't as easy as it used to be. Only 1 in 100 donors makes the cut. A decade ago, it was 1 in 10.

It's not just first-rate sperm that's in short supply. All of Israel's half a dozen or so sperm banks are scrambling to keep their liquid-nitrogen freezers stocked.

Simply put, the quality of Israeli sperm is falling at an alarming rate, and no one's sure why.

Fertility is a major issue in Israel, where memories of the Holocaust genocide are fresh, and having children is an entrenched part of Judaism. There's also a political aspect because birthrates among Arabs in Israel have at times been as much as double those of Jews, triggering a population race that some believe could one day affect who controls the land.

So the drop in the quality of sperm is raising some red flags, even though the cause remains a mystery. Theories range from the mundane (carrying cellphones in front pockets) to the far-fetched (depleted uranium from exploded munitions). Some Israeli scientists are looking at naturally occurring hormones, particularly estrogen, in Israel's water and milk. They suggest it's a mark of the country's aggressive dairy-farming methods.

In the microscope

The director of the Hadassah Sperm Bank, Ruth Har-Nir, hunches over a microscope to view a freshly donated specimen and begins to methodically count each squiggly swimmer magnified on the slide.

She is checking the quality of a prospective donor, a young graduate student hoping to earn extra cash. Though sophisticated lab machines could be used to analyze potency, Har-Nir says the old-fashioned method works best.

After a quick scan, she sits up and shakes her head. The number of spermatozoa darting around each tiny grid on the slide is two to four, well below the minimum six required, and nowhere near the 10 to 20 per grid that indicates the concentration the bank likes to see.

Also, rather than surging forward, some of the little guys flit left and right or just stall out, suggesting a weak motility.

"Under no circumstances can we accept sperm of this quality," she says. In the previous three weeks, her bank tested six candidates and rejected all. "This is the trend," she adds.

When Har-Nir helped start the sperm bank in 1991, she says, it turned away about one-third of applicants because of low quality. Using the same standard today, it would reject more than 80 percent. Though the bank relaxed its criteria, it still vetoes about two-thirds.

Long-term change

Har-Nir noticed the problem a decade ago when she began rejecting more and more sperm from otherwise healthy young men. She shared her observations with local fertility doctors, and their research has confirmed her suspicion.

In the past 10 to 15 years, the concentration of sperm samples collected by the bank dropped 37 percent from 106 million cells per milliliter to 67 million, said Dr. Ronit Haimov-Kochman, a leading Israeli infertility researcher at the Hadassah Hebrew University Medical Center.

Though declining sperm quality is an international phenomenon, the change in Israel is occurring at nearly twice the pace as other developed countries, Haimov-Kochman said. If current trends continue, she said, by 2030 the concentration of sperm from Israeli donors will drop below 20 million cells per milliliter, which many international health experts define as abnormal.

There's no evidence declining sperm quality is resulting in fewer babies. The birthrate of Israel's Jewish population has risen in recent decades, thanks largely to an increase in the number of ultraorthodox Jews, who tend to have large families.

Haimov-Kochman estimated infertility rates in Israel have risen from 10 to 15 percent in the past 15 years, but said that's in line with international trends. But she said male infertility — once believed to be the cause about half the time, just as in the U.S. — is now suspected in 70 percent of the Israeli cases.

Most worrisome, she added, is that research has focused on sperm-bank donors, mostly students who are younger and healthier than the general population.

"If this is happening to the guys on our A-team, we might only be seeing the tip of the iceberg," she said.

Banks reaching out

Sperm banks are struggling to cope. Rather than rely on walk-ins as they once did, they use marketing campaigns, posters in college sports centers and Facebook pages to attract virile candidates. The going rate for a donation has doubled in the past 10 years to about $270.

"The decline (in sperm quality) has been dramatic," Cryobank's Ronen said. "It's a shame. We see these macho, beautiful guys come to give donations, but then we're embarrassed to have to tell them that their sperm quality is so low they may actually end up coming back as a client."

He's capitalized on that, though, by offering to freeze sperm of young men with borderline quality who want to set aside a reserve in case their potency declines with age.

Har-Nir said her bank sometimes refers men with the most serious deficiencies for counseling or medical advice. But she emphasizes that rejection by the sperm bank doesn't necessarily mean they won't be able to have children naturally. It just means their sperm isn't, well, commercial quality.

Too much estrogen?

Even with the drop in sperm quality being well-documented, the cause remains unclear and the theories controversial. Some scientists fear Israelis are being overexposed to female hormones.

"People in Israel are getting quite a load of estrogen," said Laurence Shore, a retired hormone and toxicology researcher at the Kimron Veterinary Institute near Tel Aviv. "I don't think it's a good idea to expose children to such high levels of estrogen."

He said no studies have determined estrogen levels in Israel are harming humans, adding that exposure may be too low for that. But he said it might be a factor in the sperm decline.

His research has found Israeli milk and associated products such as butter and cheese can contain 10 times as much estrogen as products from other countries because of Israel's aggressive milk-production practices.

Israel is a world leader in producing milk, pumping twice as much from its cows as other parts of the world, he said. That's partly because cows are milked up to their eighth month of pregnancy, when natural estrogen levels in the milk soar, according to Shore. In nature, he said, cows usually stop giving milk to their own young when they are three months' pregnant with a new calf.

Even though many other nations have adopted similar milking practices, Shore said, Israel is one of the first and most aggressive, so it could be seeing the effect sooner.

Haimov-Kochman is looking into water quality. As a tiny nation with a shortage of water, Israel reclaims much of its used water and sewage, which is processed, used in agriculture and may find its way back into groundwater.

The water, she said, has been found to contain traces of ethinyl estradiol, a synthetic estrogen used in birth-control pills, which gets into the water through the urine of women taking the pills.

"You can't clean this from the water," she said.

Haimov-Kochman is also studying the effect of phthalates, chemicals used in plastic products that are suspected of affecting male reproductive development.

"But I can't prove any of this," she said.

Industry and government scientists dismiss fear about Israel's water and milk as unfounded, saying levels are too small to affect humans.

"Only a tiny part of the total estrogen produced by the cows ends up in the milk," said Dr. Stefan Soback, director of the Ministry of Agriculture's National Residue Control Laboratory. "It is not sufficient to determine estrogen content in milk in order to claim physiological effects to somebody that consumes it."

Batsheva Sobelman of the Los Angeles Times contributed to this report.

Some comments:

"Although estrogenic hormones in milk from nonpregnant cows are in the pg/ml range, milk from pregnant cows can contain 500 ng estradiol/l, 1 µg estrone/l (mostly as conjugated sulfate), and 10 µg progesterone/l [43] (and approximately half of dairy herd is in late pregnancy during milking). Human breast milk for infants contains little estrogen or progesterone since nursing humans are generally not pregnant. Since young children consume about 300–700 ml/day of bovine milk, they may ingest 40 to 100 ng/day of estrogen (estradiol, estrone, and estrone sulfate), and whether this can be considered a safe level is a matter of debate [45]. "

http://old.iupac.org/publications/pac/2003/pdf/7511x1859.pdf

Shore L.S. and Shemesh M. (2003). Naturally produced steroid hormones and their release into the environment. Pure and Applied Chemistry 75:1859–1871

In my opinion, 0.1 microgram per day is not a negligable dose since the daily maintanance level for post-menapausal women is 25 microgram/day. Mice will respond to 50 ng/day of estradiol (A.Kaye, personal observation). I was at the cow meeting a few years ago and some enterprizing young man wanted to extend miliking to 7 and half months. I objected strongly at the time and I trust it was never put into practice. 

Health Effects of Cows’ Milk
http://www.eps1.comlink.ne.jp/~mayus/eng/
Commercial cows’ milk has estrogenic activity as revealed
by the hypertrophic effects on the uteri of young
ovariectomized rats and immature rats
Adapted from:
Ganmaa D, Tezuka H, Enkhmaa D, Hoshi K, Sato A.
Commercial cows’ milk has uterotrophic activity on the uteri of young
ovariectomized rats and immature rats.
International Journal of Cancer 2006;118:2363-65.
Abstract
Cows’ milk has considerable amounts of estrogens, mainly in the form of estrone sulfate. To determine whether the commercial milk has any biologically significant hormonal effects, two series of uterotrophic tests were performed, one with young ovariectomized rats and the other with sexually immature rats. Thirty-six rats were used for each test. They were divided into 3 groups of 12 animals each, and were kept for 7 days on powdered chow with one of three drinking solutions: low-fat milk (experimental), artificial milk (negative control), or artificial milk containing estrone sulfate at 100 ng/ml (positive control). At autopsy, both the wet and blotted uterine weights were measured. The cell heights of uterine epithelia in ovariectomized rats were also determined. In each test, the weights of the uteri in the Low-Fat Milk group were significantly greater than those of the respective weights in the Artificial Milk group (p<0.01). Furthermore, in ovariectomized rats, the uterine epithelial-cell height in the Low-Fat Milk group was significantly greater than that observed in the Artificial Milk group (p<0.01). The uterotrophic effect of 100 ng/ml Estrone Sulfate solution was greater than that of Low-Fat Milk in immature rats (p<0.01), whereas the effect of the solution was almost comparable to that of Low-Fat Milk (p>0.05). In conclusion, commercially available low-fat milk has uterotrophic effects in both young ovariectomized rats and sexually immature rats.

 The standard in US and England is that cows are milked till 220 days or 60 dry days before calving.  Cows are pregnant the same amount of time as humans (283 days and have the same development rate, i.e. differentiation of the sexual organs occurs between 45-90 days of gestation.)