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Oregon Regional Primate Research Center Divisions of Reproductive Sciences and Neurosciences Beaverton, Oregon 97006
Address all correspondence and requests for reprints to: Cynthia L. Bethea, Ph.D., Divisions of Reproductive Sciences and Neuroscience, Oregon Regional Primate Research Center, Oregon Health and Science University, 505 NW 185th Avenue, Beaverton, Oregon 97006.
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Introduction |
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In questioning whether the mouse pituitary is like the rat pituitary, this paper highlights the "assume nothing" approach to research with genetic knockout mouse models. This issue is likely to be confronted repeatedly as we seek to confirm physiological and pharmacological observations made in other species with those made in knockout mice, but there are several other messages to take home from this observation that go beyond the actual data.
Characterization of the cell biology and physiology of the mouse has become increasingly important due to the advent of transgenic and gene knockout mice as animal models. The Turgeon and Waring laboratory has been working on the functional consequences of PR activation in rat pituitary gonadotropes, particularly related to cross-talk with GnRH receptor signaling. Thus, the availability of a PRKO mouse could allow novel extensions in this line of investigation such as teasing out pathway components, both upstream and downstream, of PR activation.
With due caution however, this study questioned whether the absence of PR (from conception) would affect the developmental sequelae in the pituitary and alter the proportion of gonadotropes in the PRKO pituitary. In addition, the critical, but basic (and often overlooked) question of whether the mouse gonadotropes resembled the rat gonadotropes with respect to PR localization and induction by E was addressed. The answers to these questions were encouraging for these investigators. That is, the phenotypic distribution of the different cell types were similar between the wild-type and the PRKO mouse. The percentages of the different pituitary cell types are similar to those observed in the rat as well (6). And, yes, E induced PR in gonadotropes in mice in the same manner as in rats. The big surprise emerged from the observation that there was a large number of other cells that contained E-induced PR; these cells turned out to be predominantly lactotropes. Why is this such a surprise? Perhaps a bit of history is called for.
From early physiological experiments, Rothchild deduced that ovarian progesterone stimulated PRL secretion in rats (7). The development of RIAs for rat and human PRL (8, 9) led to the characterization of PRL secretion during the menstrual (10) and estrous cycles (11), during pregnancy (10, 12), pseudopregnancy (13), lactation (14, 15), and in steroid replacement models (16, 17). From these and other studies, it was accepted that PRL secretion in the rat is stimulated by E (18); that PRL did not vary significantly across the human menstrual cycle (10), but that PRL increased during gestation in humans along with the greatly elevated serum concentrations of E and progesterone from the placenta (19). The increase in PRL during human gestation was initially attributed to E until the studies of Williams et al. (20). Using nonhuman primates, the Hodgen laboratory showed that E treatment alone had little effect on PRL secretion, but addition of progesterone to the E regimen significantly stimulated PRL secretion. Subsequently, studies using more frequent sampling reported higher serum PRL concentrations during the progesterone-dominated luteal phase than in the Edominated follicular phase of the human menstrual cycle (21, 22, 23). In rats, it was further demonstrated that progesterone administration could initiate, maintain, and prolong secretion of the two daily surges of PRL that characterize pseudopregnancy, the extended luteal phase of the rat (24, 25). Moreover, progesterone administration advances the proestrous surge of PRL (11) and progesterone enhances the magnitude of an E-induced proestrous-like surge of PRL in ovariectomized rats (26). Thus, the conclusion that progesterone stimulates PRL secretion was warranted.
We began to question whether progesterone acted directly at the level of the pituitary or if it acted in the hypothalamus to increase PRL secretion. At the time, it was generally accepted that E ubiquitously induced PR and then progesterone treatment decreased levels of both ER and PR. Because it was also well accepted that receptors are needed to mediate ligand-driven biological actions, it did not follow that progesterone could stimulate PRL secretion in a continuous fashion if it also eliminated its cognate receptor (as observed in the uterus).
Studies of monkey pituitary cells in culture revealed that lactotropes contain ER and they respond to E with an increase in PRL secretion, but they do not contain nuclear PR (2, 27). Only gonadotropes (LH- and FSH-producing cells) contained PR, and E treatment of cultures from long-term ovariectomized macaques directly increased the number of gonadotropes with PR (28). The same phenotypic distribution of PR was reported for the in vivo monkey pituitary (3), rat pituitary (4), and chicken pituitary (5). Hence, from 1988 until this paper, it has been widely concluded that the action of progesterone on PRL secretion is not due to an action at the level of the pituitary lactotrope. Rather, progesterone acts largely through PR in the central nervous system in rats and monkeys to increase pituitary PRL secretion although paracrine influence from neighboring gonadotropes is also present (29). Moreover, PR expression in the primate diencephalon is not severely down-regulated by progesterone in chronic paradigms as it is in the uterus (30, 31). Thus, the receptor remains present in the brain to transduce the action of the hormone.
The implication of this report seems to be that years of research on the physiology of progestin-regulated PRL secretion in rats or monkeys cannot be extrapolated to mice. This raises numerous questions that should be prioritized in times of limited resources. Should we start over and do all of the physiology again in the mouse? Do we want to know how progesterone acts at the level of the lactotrope to regulate gene expression, PRL secretion, etc. in the mouse? Or do we want to make a mouse more like a rat (or monkey) with conditional or tissue specific expression of PR in gonadotropes but not lactotropes? Does it matter? How does it matter and why? Although these questions are framed here in the context of progestin-regulated PRL secretion, there are, and will be, other examples of species diversity that may confound and frustrate, but also instruct us in nature’s perversity. I sense that some of these differences between rats and mice have come as a little more of a shock to those who use rats as a research model compared with those of us who work with primates.
Putting PRL aside for a moment, let’s take a look at the PR. This protein, which binds progestins and then acts as a gene transcription factor, plays a very important role in many aspects of reproductive function. It comes in two flavors, PR-A (the short isoform), and PR-B, which is essentially PR-A plus an extra segment on the 5' end. Genes, like people, have their preferences, and the isoforms are not functionally equivalent (32). PR in the hybrid mouse lactotrope will have gene transcriptional activity and interact with a host of coregulators. Questions immediately arise regarding which forms are used and the effect that they will have on gene expression in the mouse lactotrope.
Recent attention has focused on the differential expression, or ratios, of PR-A and -B isoforms and on variations in the expression and regulation of nuclear receptor coregulators. The mouse pituitary study (1) did not distinguish between PR isoforms, and questions remain whether mouse lactotropes and gonadotropes are lock-step in PR-A and PR-B ratios or, for that matter, whether PR isoform ratios are similar between mouse and rat gonadotropes. The authors speculate that the relative refractoriness of the mouse pituitary PR to down-regulation by progesterone compared with the rat pituitary PR (33) might relate to a difference in PR isoform ratio, but this remains to be determined. It is tempting to suggest that the mouse and the rat, representing PR-haves and have-nots in the similar context of the lactotrope, could provide models for manipulating PR form and function within a physiological framework.
In the reproductive organs, PR promotes proliferation associated with differentiation in the breast epithelium (34), and it induces terminal differentiation in the uterine endometrium (35). A great deal of work has been devoted to understanding how PR works in the context of different cells: why are physiological doses of progesterone proliferative in the breast, but antiproliferative in the uterus, and how do antiprogestins work? PR has also been implicated in the etiology of breast cancer; that is, progesterone/PR signaling can promote the growth of certain mammary tumor cells, but we still have little notion of what causes a normal cell to transform into breast cancer (36). To approach these issues, studies have compared breast and uterine cells, or normal and cancer cells, or treated and untreated cancer cell lines. When PR is expressed—A or B, good, bad, dominant, submissive, or mutated—it is in a milieu containing a potpourri of cell specific coregulators. If one examines a cell that expresses PR vs. one that does not, then generally the cell phenotype is quite different. What if we had nearly identical cells that due to a developmental cue, did or did not express PR? Could we use those cells to understand the factors that promote or repress PR gene expression, in other words, factors that influence differentiation with respect to one gene?
Hence, a larger question is what governs PR expression in mouse lactotropes. What developmental cue or cellular decision is triggered in mouse lactotropes that is different from that in monkey, rat, and chicken lactotropes? Perhaps the mouse lactotrope could be used as a naturally occurring and accessible tool to study the factors that promote or repress gene expression during development. Thus, from a cell biology point of view, it is disappointing that the mouse is not simply and conveniently a diminutive rat or primate, but the divergent strategies co-opted by the mouse also represent opportunities for investigative insight.
So, is it worth wondering whether all mice strains are the same? Clearly in many other systems they are not. Maybe other strains of mice will not have PR in lactotropes. While this line of reasoning is not particularly appealing without good evidence for support, it is worth repeating that this strain is most frequently used for transgenic studies, and we know little about other strains. If there is a mouse strain that does not express PR in lactotropes, perhaps we could use appropriate new genetic tools to compare mouse lactotropes with and without PR. We might also ask whether PR that is transfected into a lactotrope functions in a manner similar to PR that is naturally expressed in lactotropes? Or we might discover a new mechanism for cellular differentiation with regard to how PR is expressed in some cells but not others. Wait a minute, this is an issue for stem cell biologists—not endocrinologists! How did we get here from there? We were talking about this strange mouse lactotrope that expresses a nuclear transcription factor that plays a role in breast oncogenesis and all of a sudden we are wading into questions of mechanisms governing cellular differentiation that are being debated by stem cell biologists. Research on PRL will do that to you every time. You just never know where you’ll end up.
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Acknowledgments |
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Footnotes |
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Received July 26, 2001.
Accepted for publication July 27, 2001.
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References |
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This article has been cited by other articles:
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  P. M. Ismail, J. Li, F. J. DeMayo, B. W. O'Malley, and J. P. Lydon A Novel LacZ Reporter Mouse Reveals Complex Regulation of the Progesterone Receptor Promoter During Mammary Gland Development Mol. Endocrinol., November 1, 2002; 16(11): 2475 - 2489. [Abstract] [Full Text] [PDF]
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