Thesis Proposal

Louise M. Freeman

17 October 1994

My thesis examines the effects of testosterone on a sexually dimorphic neuromuscular system in rats. The spinal nucleus of the bulbocavernosus (SNB) innervates two sexually dimorphic, androgen-sensitive perineal muscles that are active during copulation: the bulbocavernosus (BC) and the levator ani (LA). Adult female rats have fewer and smaller SNB motoneurons than do males (Breedlove and Arnold, 1980; Breedlove and Arnold, 1981). Both the BC and LA are present in newborn female rats but involute almost entirely within a few days; however, a single perinatal injection of testosterone (T) can permanently save the muscles in females (Cihák, Gitmann and Hanzlíková, 1970). The survival of the muscles, by some still-unknown mechanism, spares the motoneurons from preprogrammed cell death, reducing the sex difference seen in the adult SNB (Fishman, Chism, Firestone and Breedlove, 1990). During normal male development, the testes produce a perinatal surge of androgen, which apparently acts to prevent degeneration of the BC and LA muscles and subsequently prevents the motoneuron death that occurs in females at this time.

SNB motoneurons remain sensitive to androgen into adulthood. Castration decreases and replacement testosterone increases the size of the motoneurons, the extent of their dendritic arborization, the number of synapses and gap junctions the motoneurons make and their overall excitability (Breedlove and Arnold, 1981; Kurz, Sengelaub and Arnold, 1986; Matsumoto, Arnold, Zampighi and Micevyich, 1988a; Matsumoto, Micevyich and Arnold, 1988b; Tanaka and Arnold, 1993). To understand how androgen causes such changes, it is first necessary to determine where the steroid is acting. Are these effects, like the sparing of SNB motoneurons from developmental cell death, the indirect result of androgen action on the target muscles? Or, are they a direct effect of androgen on the motoneurons themselves? Both SNB motoneurons and BC/LA muscles express the androgen receptor (AR) during adulthood, so both are potential sites of action.

By breeding animals that are genetic mosaics for the AR gene, I have designed a model for testing the hypothesis that androgen acts directly on the motoneurons themselves to increase soma size in adulthood. The testicular feminization mutation (Tfm) is a sex-linked recessive mutation that causes a single amino acid substitution in the rat AR (Yarbrough, Quarmby, Simental, Joseph, Sart, Lubahn, Olsen, French and Wilson, 1990). Affected males possess functional testes and normal circulating levels of testosterone, but because only 10-15% of the mutant ARs bind testosterone, the rats are insensitive to androgens and appear phenotypically female (Stanley, Gumbreck and Allison, 1973; Wilson, Griffin, Leshin and MacDonald, 1983). Females heterozygous for this gene are carriers of the trait but are themselves unaffected. If a carrier pup is given perinatal testosterone, the BC/LA muscles can be saved. However, because of random X chromosome inactivation early in development, not all of her SNB cells will be capable of synthesizing a functional AR. Immunocytochemistry using an antibody against the AR can distinguish between SNB motoneurons expressing the mutant receptor and those expressing the wild-type receptor. Since ligand-binding is apparently necessary for nuclear translocation of the AR, wild-type motoneurons show nuclear immunostaining while Tfm motoneurons do not. Thus, the two different types of motoneurons (androgen-sensitive and androgen-insensitive) can be detected side-by-side in the spinal cord and compared in a within-subjects design.

By giving such mosaic females long-term testosterone treatment in adulthood and comparing the size of Tfm and wild-type motoneurons, I hope to determine whether androgen acts directly on the motoneuron to increase cell size. If so, the wild-type cells should be larger than the Tfm cells in that animal. If androgen indirectly increases cell size by acting somewhere else, for instance, at the target muscle, the Tfm and wild-type motoneurons should be the same size. In addition to addressing this site of action questions, this model may also prove useful in investigating which effects of adult testosterone on SNB cells are dependent on a functional AR and which genes are co-expressed with, and therefore possibly regulated by, the AR in these motoneurons.

Experiment 1: Breeding of animals who are genetic mosaics for the androgen receptor

The AR gene is carried on the X chromosome. As is the case with other mammalian sex-linked genes, females (XX) have two copies and males (XY) only one. In females, one X chromosome per cell condenses into a Barr body early in development; such condensation is apparently random. No such condensation occurs in males (Fig. 1). Males with the Tfm trait (XTfm Y) are entirely androgen-insensitive (Fig 1a) and externally appear to be female, despite abdominal testes and normal-to-elevated circulating levels of testosterone (Stanley et al., 1973; Wilson et al., 1983). In female carriers of the Tfm trait (XTfm X), only those cells in which the X-chromosome with the wild-type gene remains uncondensed can express a functional AR (Fig 1b). As a result, in adult tissues whose survival is not influenced directly by developmental androgens, half the cells are androgen insensitive. Normally, carrier females are phenotypically indistinguishable from their wild-type sisters.

Carriers are normally identified by breeding them and identifying affected sons. However, this is not possible in female rats who have been given perinatal androgen to spare the SNB system, since such treatment renders the females anovulatory. Instead, I identified carriers by giving an earlier androgen treatment and examining the nipples, which are influenced by androgen during development. Nipples are sexually dimorphic in rats: prenatal androgen normally prevents nipple formation in males, while absence of androgen permits formation in females. Treating a pregnant dam with androgen on days E16-20 eliminates nipple formation in her female pups (Goldman, Shapiro and Neuman, 1976).

Method

Known carriers (identified by having Tfm-affected male offspring in previous litters) were paired with males, and daily vaginal smears were examined for the presence of sperm. The morning of the first positive sperm was recorded as E1. On days E16-20, each dam was injected subcutaneously with 2 mg testosterone propionate (TP) dissolved in sesame oil. Some litters were born normally (E23), but approximately 2/3's of the dams failed to deliver the pups by E23 (an unexpected side effect of the testosterone injections). In that case, the dam was anesthetized with ether, and the pups delivered by cesarian section and fostered to another lactating dam. Each pup was given TP on postnatal days 1 and 3 (1 mg in sesame oil, s.c.) to save the SNB system (Fig 2).

Results

By P30, four distinct phenotypes were distinguishable among the pups, 3 of which are illustrated in Fig. 3. Wild-type males had a clearly developed scrotal sac with testes and no nipples. Tfm males had palpable abdominal testes and nipples (Fig 3a). Among animals with no palpable testes (females) some had no visible nipples and clearly masculinized external genitalia; these animals are presumably 100% androgen sensitive non-carriers (XX, Fig 3b). However, other females had a full array of nipples and only partially masculinized external genitalia (Fig. 3c). Since these females have nipples, they must have some androgen-insensitive tissue; therefore, they must be carriers of the Tfm mutation.

Experiment 2: Distinguishing Tfm from wild-type SNB motoneurons by immunocytochemistry

Methods

I used immunocytochemistry (ICC) to detect AR in the SNB of wild-type males, Tfm-affected males and long-term castrate males with and without hormone replacement therapy. The primary antisera used was PG21 (provided by G. Prins), a polyclonal rabbit antibody generated against the first 21 animo acids of the rat AR, a portion of the protein involved neither in ligand nor DNA-binding (Jenster, van der Korput, van Vanvroonhoven, van der Vanderkwast, Trapman and Brinkmann, 1991). Animals formed five experimental groups: 1) intact males, 2) long-term castrate males, long-term castrate males who received androgen either 3) 30 min (2 mg T intravenously) or 4) 8 hrs (2 mg TP, subcutaneously) before sacrifice and 5) affected Tfm males. The long-term castrate males were gonadectomized (GNX) under ketamine cocktail anesthesia (60.6 mg ketamine, 6.06 mg xylazine, 0.91 mg acepromazine/ml; 0.09 ml/100 g body weight, i.p.) 4-5 weeks before sacrifice.

All animals received a lethal dose of sodium pentobarbital and were perfused intracardially with physiological saline followed by 4% paraformaldehyde. Spinal cords were removed, postfixed 1-3 hrs, then soaked overnight in 20% buffered sucrose. The following day the lumbosacral segments were frozen-sectioned in the frontal plane at 50 um, and the free-floating sections rinsed in a phosphate-buffered gelatin triton solution (PGT, 0.1 M phosphate buffer with 0.1% gelatin and 0.3% triton added).

Sections were incubated first for 1 hr at room temperature in 10% normal goat serum (NGS), then for 48 hr at 4deg.C in 0.6 ug/ml solution of PG21 in PGT with 4% NGS. Sections were rinsed 3 X 5 min in PGT prior to reaction with an avidin-biotin-peroxidase complex kit from Vector Laboratories (Burlingame, CA). Following a 1 hr incubation in biotinylated 2deg. antibody (goat anti-rabbit), tissue was rinsed in PGT, then incubated for 1 hr in avidin-biotin complex. Staining was visualized with H2O2 and diaminobenzidine and intensified with 1% NiCl2. Sections were mounted on gelatin-subbed slides, half of which were counterstained with neutral red and coverslipped with Permount. The SNB was identified in counterstained tissue under light microscopy and the percentage of cells in each motoneuron pool showing a nuclear reaction product was determined. All counts were made by an observer unaware of treatment group.

Specificity of staining was confirmed by three series of control experiments which A) the 1deg. antibody was omitted, or B) was preabsorbed for 30 min with 20 X excess of the AR peptide used to generate the antibody, or C) was preabsorbed for 30 min with 20 X excess of an AR peptide distant to the peptide used for immunization. Sections were processed and counterstained as before.

Results and Interpretations

In intact wild-type males, most of the motoneurons in the SNB displayed a dark nuclear accumulation of reaction product (Fig. 4a). Elimination of the primary antibody or preabsorption with the immunizing peptide eliminated this nuclear immunostaining (Fig. 4b,c), while preabsorption with the distant AR peptide did not (Fig. 4d), indicating the specificity of this antisera for the immunizing epitopes found in the AR in the present tissue.

The androgen status of the animal markedly altered the pattern of immunostaining in the spinal motoneurons. I saw little or no nuclear immunostaining in any group of motoneurons from GNX or Tfm males. (Fig. 5) Animals in these two groups showed some apparent cytoplasmic staining in SNB cells, most easily visualized in non-counterstained tissue (Fig. 6a), and such cytoplasmic staining was not evident in the intact male SNB cells that exhibited nuclear staining (Figure 6c). TP replacement injections of GNX males 8 hrs before sacrifice restored nuclear immunostaining to levels slightly higher than that of intact animals in all three cell groups, while animals exposed to T 30 min before sacrifice showed levels of immunostaining comparable to intacts. The detection of apparent cytoplasmic AR in GNX males and restoration of SNB nuclear immunostaining to intact levels after only 30 min of T exposure suggest that the AR protein is present in those motoneurons even in the long-term absence of androgen. The relative lack of cytoplasmic immunostaining in non-counterstained motoneurons of intact males (Fig. 6c), coupled with the rapid increase in nuclear labeling with T replacement in castrates suggests ligand-activated nuclear translocation of preexisting receptor.

The low level of nuclear staining seen in Tfm males can therefore be explained by the small percentage of Tfm receptors that bind T; although the Tfm rat AR gene is transcribed at the same rate as the wild type gene (Yarbrough et al., 1990), there is little translocation because there is little ligand-binding. Yarbrough et al.,(1990) found that the Tfm pituitary shows nuclear immunocytochemical staining only at super-physiological doses of T, suggesting that the low affinity binding of T shown by most mutant receptor proteins prevents nuclear translocation except at very high levels of hormone or that all of the few functional receptors must be recruited to reach detectable levels. In accordance, in a pilot study where I gave gonadally intact Tfm rats supraphysiological replacement androgen (three 2 mg injections of TP in the 24 hours prior to sacrifice) had a notable amount of nuclear immunostaining the SNB. These results indicate that PG21 ICC in the presence of an intermediate, physiological level of systemic androgen can distinguish Tfm from wild-type motoneurons in the rat SNB.

An alternative interpretation of the results is that PG21 binds preferentially to ligand-bound receptor, in which case the unoccupied receptors in the castrate and Tfm rats might be nuclear but remain undetected. This alternative explanation does not preclude the use of PG21 ICC as a tool for distinguishing Tfm from wild-type motoneurons.

Experiment 3: AR-ICC in Tfm carriers

The carriers generated in Exp. 1 were randomly assigned to one of two groups. Under metaphane anesthesia, one group (N=6) received 2 20mm testosterone-filled Silastic capsules; the other received empty capsules. Capsules were left in place 4-6 weeks months, then removed 24 hours prior to sacrifice (Fig. 7). Four wild-type sisters of mosaics and four Tfm affected brothers served as controls. The WT females received no hormone treatment in adulthood, while the Tfm males were anesthetized with ketamine and castrated 24 hours prior to sacrifice.

Three hours prior to sacrifice, all animals received a 1ug/g body weight injection of TP so that androgen receptors could be visualized by immunocytochemistry. Animals were overdosed, perfused and processed with the AR-ICC procedure of Exp 2. All tissues were coverslipped without counterstain and the number of immunolabelled nuclei counted and mapped. Coverslips were then soaked off and the tissue counterstained with neutral red Nissl stain and recoverslipped. The total number of SNB cells was then counted and the percentage of labelled cells calculated. All counts were made without knowledge of treatment group.

In the mosaic animals, the soma and nuclei of 12 AR+ and 12 AR- were traced with a camera lucida and the area measured with a digitizing pad and microcomputer. Drawings and measurements were taken without knowledge of adult hormone treatment. Soma size data were evalulated by 2-way ANOVA (treatment by AR status) with AR status as a within-subjects measure (Fig 9).

Results

The cell numbers and percentages of immunolabelled cells are summarized in Table 1.

	Table 1- SNB cell number ± SEM
	# nuclear labelled	total SNB cell #	% labelled SNB cells
WT female	84 ± 19.7	118.5 ± 12.1	69 ± 12.7
Mosaics	42 ± 5.2	85 ± 6.5	49 ± 3.7
Tfm male	15 ± 5.9	79 ± 8.3	18 ± 6.5

As expected, the wt females had the highest percentage of immunolabelled SNB cells and the Tfm males the least, with the mosaics intermediate. Importantly, both wild-type and Tfm motoneurons were detectable in the SNB of mosaics (Fig. 8). However, the percentage of labelled SNB cells was lower in the WT females than in the males of Exp. 2 and the percentage of labelled SNB cells in the Tfm was higher. Reasons for this discrepancy may include the use of a different batch of primary antisera and the lower TP injection given to visualize androgen receptors. As a consequence, this methodology is only around 70% accurate in distinguishing Tfm from wild-type motoneurons.

Also unexpected was the failure of the early T treatments to save a significant number of SNB cells in the mosaics. Two factors may account for this: 1) the mosaics would have, on average, half the amount of androgen-sensitive muscle tissue as the WT females. As a consequence, LA muscles in mosaics are considerably smaller than in the WT females. If androgen acts on the target muscle to indirectly spare the motoneurons from cell death, it has a smaller target to act on in the mosaics. 2) Most dams delivered the pups 24 hours later than expected, meaning the P1 injection was actually not given until P2. This is late in the critical period for sparing SNB cells (Breedlove and Arnold, 1983), so, while the treatment may have been sufficient in WT females, it was insufficient in mosaic females, who had a smaller target muscle.

Despite these limitations, the results of the soma size measurements is encouraging (Fig. 10). There is a main effect of AR status; labelled by AR-ICC are significantly larger than those that are not. The difference is larger in the group receiving T capsules, although the interaction does not reach significance. There are several explanations for the WT motoneurons being larger in animals that do not receive T in adulthood. 1) Androgen acted on the individual motoneuron during development to permanently increase its size. Early androgen treatments can permanently increase SNB motoneuron size, but the site of action for this effect is not known (Forger, Fishman and Breedlove, 1992). Steroid autoradiography has failed to detect AR expression in SNB motoneurons before P3 (Jordan, Breedlove and Arnold, 1991), but it is possible that large doses on androgen at that time could induce expression. 2) A subset of SNB motoneurons innervate the external anal sphincter. These motoneurons are smaller than those innervating the BC and LA muscles and are reported not to increase their size in response to adult steroid treatment (Collins, Seymour and Klugewicz, 1992). These motoneurons may not be as likely to express AR, and could be overrepresented in the AR- population. 3) Androgens from another source, such as the adrenals, may be having a small effect on SNB cell size. Whatever the case, I am interested in knowing whether there is an additional effect of adult T on the AR+ SNB motoneurons.

Two things appear necessary to use this model to address the question of site of adult androgen action on SNB cells. First, it would be desirable to increase the number of SNB cells in mosaics. This can probably be achieved by extending the prenatal hormone treatment through E22 to include more of the SNB critical period and by delivering by c-section early on day 23 and beginning T treatments immediately. Second, the accuracy of the AR-ICC procedure in distinguishing Tfm from wild-type motoneurons must be increased. I believe this can be accomplished by using the anti-steroid hydroxyflutamide (OHF) instead of TP to visualize AR in the ICC procedure.

Experiment 4: Is hydroxyflutamide as effective as T in distinguishing Tfm from wild-type motoneurons? (in progress)

One reason for the higher than expected percentage of immunolabelled cells in the Tfm males is that the bolus of injected T activated the 10-15% of functional AR and upregulated AR protein expression, causing there to be enough functional receptor present in some motoneurons to reach the threshold for a positive signal. This problem could be eliminated by using a anti-steroid such as hydroxyflutamide (OHF) which binds the receptor and causes nuclear translocation but does not activate gene transcription (Wong, Zhou, Sar and Wilson, 1994). hydroxyflutamide (OHF). The following study is designed to determine if AR-ICC following an OHF injection is any more effective at distinguishing WT from Tfm motoneurons.

Animals form 4 groups: WT male,

GNX male + 2mg TP, 8 hrs before sac

GNX male + 2mg OHF, 8 hrs before sac

GNX Tfm male + 2mg OHF, 8 hrs before sac

Animals are sacrificed and processed with immunocytochemistry as before, numbers of immunolabelled and total SNB motoneurons are counted and the percentage of immunolabelling calculated.

Results are preliminary, but in the animals examined so far it looks like OHF is as effective as T. I see 90% label in WT males with <5% label in Tfm. In inability of OHF to activate gene transcription may make it a better tool for distinguishing Tfm from wild-type motoneurons with AR-ICC; the anti-steroid would cause translocation of the receptor, but would not induce enough additional AR to cause a positive signal in Tfm motoneurons. Alternately, OHF could have even less affinity for Tfm receptor than T, or the Tfm receptor, when bound to OHF, could undergo a conformational change that causes it to have less affinity for antisera. As in Exp. 3, the mechanism is irrelevant to AR-ICC's use as a tool. An added advantage is that a larger dose (2 mg) of OHF can be used without the nuclear immunolabelling seen in Tfm SNB cells at supraphysiological levels of T. This higher dose produces darker immunostaining than the near-physiological T doses used in Exp. 3, making the nuclei easier to detect in counterstained tissue. This may make it possible to count the motoneurons in counterstained tissue, as was the case in Exp. 2.

Experiment 5 (proposed)

Methods are the same as in Exp. 3, with the following modifications (Fig. 10):

1) Prenatal TP treatments will be increased to 2 mg/day for E17-E22.

2) Pups will be delivered by c-section on E23. Pups will get 1 mg TP on days P1-P3

3) 2 mg OHF injections will be given to all animals 8 hours before sacrifice, rather than the 1 ug/g body weight 3 hours before sacrifice.

4) number of AR+ and AR- cells drawn per animal will be increased from 12 to 20.

I am optimistic that those modifications should sufficiently increase the power of the study to the point where I can tell if the trends seen in Exp. 3 reflect real differences.

Possible outcomes and interpretations:

1) AR+ cells will be larger than AR- only in animals receiving long-term T-treatment in adulthood.

Interpretation: Adult androgen acts directly on the motoneuron to increase cell size.

2) SNB cells in the T-capsule group will be larger than in blanks, regardless of AR status.

Interpretation: Adult androgen acts at some other site, possibly the target muscle or supraspinal afferents to indirectly increase cell size.

3) AR+ cells are larger than AR- in both T capsule and blank groups.

Interpretation: Any direct effect of androgens on motoneuron size occurs during development; adult androgen treatment has no additional effect.

4) AR+ cells are larger in both groups, but the size difference is significantly greater in the T capsule group.

Interpretation: Androgen can act both during development and adulthood to directly increase SNB soma size.

References

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