By David N. Leff

The Greeks had a word for it, all right ¿ oistros ¿ meaning ¿frenzy¿ or ¿mad desire.¿ These terms describe the mental and physical sexual arousal of a female mammal desirous of mating ¿ ¿estrus¿ in English. But not to worry; human females are virtually the only mammals excused from estrus.

In lesser animals, this condition of being in heat kicks in immediately preceding ovulation, a point of maximum receptivity to copulation. Whence the word for the hormone estrogen (estrus plus gen - Greek for ¿born.¿)

¿We¿ve always known that estrogen is very important to all aspects of female fertility,¿ observed molecular biologist John Couse, at the National Institute of Environmental Health Sciences (NIEHS) in Triangle Park, N.C. The hormone is produced in the ovary,¿ he went on, ¿but it was never really thought that estrogen had a major role in the ovaries. Its major job was in preparing the uterus for pregnancy.¿

That politically correct common knowledge has turned out to be lacking, thanks to three strains of knockout mice generated at NIEHS in the past seven years. Another facet of this received wisdom held that just one type of the estrogen hormone circulated in the human body. ¿Prior to 95 and 96,¿ Couse pointed out, ¿it was thought that only a single estrogen receptor existed. And a receptor,¿ he explained, ¿is what allows a cell to respond to the hormone, which flows throughout the whole body. So it¿s going to come in contact with all cells, but some cells are going to ignore it, while others are going to respond to the estrogen because they have reproductive function.¿

In 1993, scientists at the NIEHS knocked out that only-known estrogen receptor gene from fetal mice. ¿When they set out to do that,¿ Couse recounted, ¿there was a belief in the estrogen-research field that it was impossible to do. That is, the animal would die before it was even born because estrogen was so important. But we actually were able to generate an animal that was missing this receptor. These mice,¿ he added, ¿had gonadal and behavioral abnormalities, resulting in infertility in both females and males ¿ but they didn¿t die.¿

A Second Receptor Pops Up

He continued: ¿Then, in 1996, came the discovery of a second, separate, estrogen receptor. That was a surprise. In fact, the people who cloned it were not really looking for it. It made us think that maybe this was why we got viable animals the first time, because this second receptor¿s estrogen allowed it.¿

Now with two estrogen receptors (ER) instead of one, workers in the field named them alpha ER and beta ER. Couse¿s colleagues duly knocked the beta ER gene out of a second string of mice. ¿The phenotypes in that beta animal,¿ he recalled, ¿were a little bit harder to describe, simply because in going into the first animal, the alpha knockout, we had 20 or 30 years of research behind us as to what this receptor . . .did. So we could predict what would happen if we lost the receptor.

¿Those beta mice turned out to be not so dramatically affected as the alpha knockouts; while the males are fertile, the females are subfertile, due to inefficient ovarian function.¿

In each of these two mouse strains, while one estrogen receptor is absent, the second one is still present, so the two are presumably exchanging cross-talk, providing sufficient hormone signaling to preserve life. ¿The one animal missing alpha,¿ Couse elaborated, ¿still had the beta receptor, and vice versa.¿ So the group set out to generate a double knockout.

Couse is lead author of a paper in today¿s Science, dated Dec. 17, 1999, and titled: ¿Postnatal sex reversal of the ovaries in mice lacking estrogen receptors a and b¿

¿So now we have three model knockouts,¿ Couse said, ¿one animal missing ER-alpha, a second lacking ER-beta and a third ¿ a double-knockout - missing both. Making the double was really the easy part.¿

He explained: ¿For the individual-gene mice, the alpha and beta estrogen receptors, we used traditional knockout technology. Then, to generate a double, we bred animals that are heterozygous for those mutations, that is, mice that are carriers of the functional deletion. They have at least two copies of every gene - one that was disrupted and one that was normal. These mice were fertile; they didn¿t show any of the abnormalities because they still had one gene copy. But they could pass the mutation on to their progeny.

¿This yielded one homozygous double-knockout in every 16 births, and as their sex ration was a normal 50:50, to get a female, the odds were 1 in 32. We¿ve generated close to 500 mice through this breeding scheme just to get our hands on these knockouts.¿

Sex Reversal, Cellular Style

As for the sex-reversal aspect, Couse commented, ¿That term is a little bit misleading. It¿s a nice term for a Science paper title because the double animals look like females on the outside. It¿s not until you get a look at the ovary that you actually see this sex reversal. What it amounts to is we are seeing a Sertoli cell in the ovary of an adult female double knockout ¿ a cell ordinarily found only in the testis of a male.

¿It appears,¿ Couse said, ¿that the uniqueness of our model is two-fold: One, we¿re the first to show that this could happen by disrupting estrogen signaling. And second - at least in the mouse - it seems that we¿re the first to show that this could happen in the adult. Others have found it necessary to use a fetal ovary precursor. In fact, some investigators in the past said that if you take an ovary from an older animal to try to induce this effect, it won¿t happen. They put forth that there¿s a window of susceptibility where under the right conditions you can cause it to form the male-like testicular cells. And if you miss that window, it¿s locked in to be an ovary always.