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Biology of Reproduction 60, 119-127 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Development and Differentiation of the Interstitial and Tubular Compartments of Fetal Porcine Testes1

M.A. Kaminski3,a, C.J. Corbinb, and A.J. Conley2,b

a Department of Animal and Range Science, North Dakota State University, Fargo, North Dakota 58105 b Department of Population Health and Reproduction, University of California, Davis, California 95616


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Male differentiation is initiated by fetal testicular androgen synthesis, catalyzed by the enzyme 17{alpha}-hydroxylase/17,20 lyase cytochrome P450 (P450c17). This study was an investigation of testicular development and differentiation in porcine fetuses recovered on Days 30–42 of gestation. The expression of P450c17 was localized in fetal gonads by in situ hybridization and immunocytochemistry and related to cellular proliferation through expression of the proliferating cell nuclear antigen (PCNA). Gonadal P450c17 expression was quantified by Western immunoblot analysis and related to testosterone secretion by cultured explants of fetal gonads. P450c17 transcripts were detected in the interstitium surrounding testicular cords preceding the appearance of the enzyme protein. The intensity of both P450c17 hybridization and staining was greater in Yorkshire fetal gonads, which also exhibited more advanced tubular development. PCNA staining was prominent within tubular primordia and was higher in testes from Yorkshire than from Meishan fetuses on all days examined. P450c17 expression paralleled testosterone secretion, which decreased by Day 42, and was generally less in cultures of Meishan than of Yorkshire fetal gonads. These data demonstrate that the expression of P450c17 in porcine fetal testes coincides with differentiation of central medullary cells and androgen secretion during gonadal development between Days 30 and 42 of gestation. This occurs as medullary cords organize and is associated with changes in cellular proliferation within the tubular compartment.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gonadogenesis forms the foundation of all subsequent sexual differentiation in mammals regardless of the sex chromosome constitution. Each mammalian fetus has the potential to develop both male and female reproductive systems, and normal sexual development requires one duct system to differentiate as the other regresses [1]. The stimulus for the differentiation of the male (wolffian) ducts, and simultaneous regression of the female (Müllerian) ducts, involves the synthesis and secretion of androgens and other hormones by the embryonic testes [2]. In the absence of fetal testicular endocrine function [3], or the ability to respond to hormones secreted by the testis [4], the primordia of the male tract degenerate and those of the female system become stabilized. Hence, male sexual differentiation is an active process, which is initiated before ovarian development begins in females, and is dependent on fetal testicular differentiation and growth.

The synthesis of testosterone, the principal androgen secreted by the fetal testes, is catalyzed by the enzyme 17{alpha}-hydroxylase/17,20-lyase cytochrome P450 (P450c17; [5]). Consistent with androgen production, P450c17 is expressed early during testes development in pigs [6]. It might be presumed that the Leydig cell is the site of P450c17 expression in the fetal testes as it is in the newborn pig [7] and mature boar [8]. However, P450c17 expression has been reported in several unexpected sites including the liver [9] and the stomach [10]. Moreover, the site of P450 expression can change with development. P450c17 is expressed in the fetal adrenal gland but not in the mature adrenal gland of the mouse [11], for instance, and the expression of another steroidogenic cytochrome P450, aromatase, changes from the Sertoli cell to the Leydig cell during the transition from fetal to mature function in male rats [12]. Clearly, P450 enzyme expression is not restricted to classical steroidogenic cells, and it may be regulated very differently in porcine conceptus than in adult tissues [13]. Moreover, the functional relevance of recently identified transcription factors to Leydig cell differentiation, currently based on a temporal association with gonadogenesis [14, 15], depends entirely on expression in the same cell. Unfortunately, definitive evidence on this point is lacking. In fact, only a single report, of studies that were conducted in the rat [16], appears to document the cellular site of P450c17 during mammalian testicular development despite excellent morphological documentation of gonadogenesis in several species and numerous reports on steroid secretion [1]. Therefore, determining the exact spatial location of expression of P450c17 in the porcine fetal testes is an important first step in investigating the control of testicular androgen secretion, Leydig cell differentiation, and male sexual development.

The present study was conducted to test the hypothesis that the functional endocrine development of the porcine fetal testes would be associated with the appearance of P450c17, and that expression of this enzyme would be restricted to the progenitors of the Leydig cells in the testicular interstitium. Though experiments were conducted principally using litters from Yorkshire dams, additional litters from Chinese Meishan dams were also examined to see whether there was evidence of possible differences between breeds in the timing of these events. Recent studies conducted in this laboratory have demonstrated the existence of developmental differences in the expression of steroidogenic enzymes in Yorkshire and Meishan conceptuses at the preattachment, blastocyst stage [17].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Embryos were collected from domestic Yorkshire and Chinese Meishan sows on Days 30, 34, 38, or 42 of gestation after slaughter in a USDA-inspected facility, as approved by the Institutional Animal Care and Use Committee. The reproductive tract of each sow was excised within 20–30 min of slaughter and immediately placed on ice for transport to the laboratory. At the laboratory, the uterine horns were dissected free of the mesometrium. Starting at the left tip, each uterine horn was opened longitudinally along the mesometrial border, care being taken not to disturb placental attachments. The chorioallantoic membrane was punctured as each conceptus protruded, the allantoic fluid collected into a graduated cylinder, and the volume recorded. After the weights of chorioallantoic membranes and fetuses were recorded, each gonad was dissected out. One gonad from each pair was immediately fixed in 4% paraformaldehyde and embedded in paraffin, sectioned, and stained with hematoxylin and eosin for identification of fetal sex as well as for immunocytochemistry and in situ hybridization. The second gonad was used for explant culture to evaluate androgen synthesis. After 6 h of culture, gonads were frozen and stored at -80°C. Culture medium and allantoic fluid were frozen at -80°C and assayed later for testosterone.

Immunocytochemistry (ICC)

The procedures used for ICC were similar to those described previously for proliferating cell nuclear antigen (PCNA) and P450c17 [18, 19]. Sections were incubated in a humid (100%) chamber at 60°C for 10 min, deparaffinized, rehydrated, and incubated with blocking buffer for 20 min. Sections were incubated for 1.5 h at room temperature with primary antibody. PCNA was immunolocalized by means of a specific monoclonal antibody (Boehringer Mannheim, Indianapolis, IN) diluted 1:500 in blocking buffer, whereas anti-P450c17 (courtesy of Dr. Anita Payne, Stanford University, Palo Alto, CA) was diluted 1:1000 in blocking buffer. Control sections were incubated with normal serum in the place of primary antibody. After incubation with primary antibody for 1 h, sections were subjected to two 5-min washes in PBT (50 mM phosphate, 150 mM NaCl, Triton X-100, pH 7.6). The primary antibody was detected with the aid of a biotinylated secondary antibody (horse anti-mouse IgG, PCNA; goat anti-rabbit IgG, P450c17) and avidin-biotin peroxidase conjugate along with 3,3'-diaminobenzidine as the substrate, all from Vector Labs (Burlingame, CA).

In Situ Hybridization (ISH)

Polymerase chain reaction was used to amplify 640 base pairs (bp) of DNA encoding the 5' end of the porcine P450c17 testes cDNA, which was subsequently subcloned into pGEM-T (Promega, Madison, WI). After linearization with SacII, the radiolabeled antisense cRNA probe was transcribed in vitro with Sp6 RNA polymerase (Stratagene, La Jolla, CA) and [35S]uridine triphosphate (Amersham, Arlington Heights, IL). The control sense cRNA probe was generated with T7 RNA polymerase after template linearization with SalI.

Hybridization was performed according to the established protocol of Keeney et al. [11]. Porcine embryonic gonads from all stages were fixed for 24 h in 4% paraformaldehyde (in RNase-free PBS, pH 7) and embedded in paraffin. A pair of serial sections, 5 µm thick, were dried on ProbeOn Plus slides (Fisher Scientific, Santa Clara, CA). Prehybridized sections were deparaffinized and rehydrated; they were then treated with 4% paraformaldehyde, pronase E (protease type XIV; 110 mg/ml), and triethanolamine (0.1 M, pH 8.0) containing acetic anhydride. Separate hybridization mixtures of antisense and sense [35S]uridine triphosphate-labeled cRNA probes were applied to sections on one or the other half of each slide. After overnight hybridization at 50°C, sections were subjected to high-stringency washes and digestion with RNase A (20 µg/ml). Sections were finally dehydrated, air dried, and dipped in Kodak NTB-2 nuclear track emulsion (Eastman Kodak, Rochester, NY). Slides were exposed for 3 days, developed photographically, and stained with hematoxylin and eosin.

Western Analysis

Individual gonads were sonicated in PBS containing 1% sodium cholate and 0.1% SDS. The protein concentration for each sample was estimated using the BCA Protein Assay Reagent (Pierce Chemical Co., Rockford, IL). Homogenates containing 25 mg of protein were subjected to electrophoresis on 8% SDS-PAGE gels [6, 19, 20]. Separated proteins were electroblotted onto nitrocellulose membranes and immunoblotted with anti-porcine P450c17 antisera (1:2000). A chemiluminescent detection system was used as recommended (Amersham). Autoradiograms were quantified by densitometry.

Tissue Culture

Individual fetal gonads were cultured as described by Conley et al. [6]. Briefly, gonads were suspended on stainless steel grids covered with lens paper over Dulbecco's Modified Eagle's medium (Gibco-BRL, Gaithersberg, MD) with 1% fetal bovine serum. Incubation was carried out for 6 h at 37°C in 5% CO2. At the conclusion of culture, the medium was collected, frozen, and stored at -20°C until assayed for testosterone.

RIAs

Testosterone was quantified in 50 µl of unextracted culture medium and allantoic fluid in separate assays using a Coat-A-Count total testosterone kit (Diagnostic Products Corporation, Springfield, IL). Sensitivity of the assay was 200 pg/ml, and the intraassay coefficient of variation was 9.9% based on sample pools (1.21 ± 0.08 ng/ml, n = 8).

Statistical Analysis

Numeric data from densitometry of autoradiograms resulting from Western immunoblots and testosterone secretion by cultured fetal testes were analyzed by ANOVA using the General Linear Models procedure, and correlation coefficients were calculated, using SAS (Statistical Analysis System Institute, Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Of the 138 conceptuses collected from Day 30 to 42 of gestation, 52.8% on the basis on gonadal histomorphology were male. Male and female gonads were easily differentiated from one another on all days examined. Ovaries were typified by the presence of a cuboidal or columnar germinal epithelium surrounding a poorly organized stroma, whereas the testes exhibited a thin, almost squamous surface epithelium and well-defined medullary cords. Beneath the surface epithelium of the testes were several cell layers of undifferentiated mesenchyme and interstitium separating the organizing testicular cords (data not shown).

The differentiation of fetal Leydig cells was evidenced by the cytoplasmic localization of immunodetectable P450c17 (Fig. 1). Leydig cells in the medullary region of the developing testes were the first to express this enzyme, which was observed on Day 30 of gestation in Yorkshire fetuses (Fig. 1B) but was not detectable in Meishan fetuses until Day 34 (Fig. 1, A vs. C). Both the amount and intensity of positive staining for P450c17 increased thereafter in the Yorkshire fetal testes, reaching a peak by Day 38 and then declining somewhat on Day 42 (Fig. 1, D, F, and H). P450c17 staining also increased after Day 34 in Meishan fetal testes, but no obvious decline in expression was seen by Day 42 (Fig. 1, E and F). In general, the pattern and level of P450c17 staining seen in Meishan fetal testes were consistent with a delay in Leydig cell differentiation of as much as possibly 4 days in comparison to Yorkshire testes development.



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  		FIG. 1. Immunocytochemical localization of P450c17 in sections of porcine fetal testes recovered on Days 30 (A, B), 34 (C, D), 38 (E, F), and 42 (G, H) of gestation from Meishan (left panels) and Yorkshire (right panels) dams. Single isolated Leydig cells (arrow) located within the interstitium (I) stained intensely for P450c17, whereas no staining was detected within testicular cords (C). Bar = 30 µm; all micrographs were taken at the same magnification.

The localization of P450c17 transcripts by ISH affirmed the results obtained by ICC (Fig. 2). Low levels of transcript appeared in the medullary regions of the Yorkshire fetal testes on Day 30 of gestation (Fig. 2A). The most intense level of hybridization for P450c17 was observed in the differentiating interstitium of Yorkshire testes on Days 34 and 38 (Fig. 2, B–E), decreasing markedly by Day 42 (Fig. 2F). As was the case with ICC, ISH was unable to detect the presence of P450c17 expression in Day 30 Meishan testes. Expression of P450c17 in Day 34 Meishan testes (Fig. 2B) exhibited the same trend as was observed for the protein, with the levels of expression lagging behind those of Yorkshires on the same day of gestation. At no time did the level of P450c17 transcript in Meishan testes equal the peak level of expression observed in Day 34 Yorkshire testes (Fig. 2). No hybridization was observed with the sense control P450c17 probe. Similarly, no evidence of P450c17 expression was observed in cells within the cords by ICC or ISH in either Yorkshire or Meishan testes. Additionally, sections of fetal ovaries lacked discernible staining for P450c17 at any stage examined with the exception of those from one female Yorkshire fetus collected at Day 42 of development. Interestingly, positive immunostaining for P450c17 was observed in ovarian sections from this fetus in several isolated cells that were located within the central medullary region of the gonad (data not shown).



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  		FIG. 2. In situ hybridization of P450c17 in sections of porcine fetal testes recovered on Days 30 (A), 34 (B, C), 38 (D, E), and 42 (F) of gestation from Yorkshire (A, C, E, F) and Meishan (B, D) dams. Original magnification using x10 objective.

Further confirmation of the developmental expression of P450c17 in fetal gonads from Day 30 to 42 of gestation was sought using Western immunoblot analysis. A single band, 51–53 kDa in size, was observed in Yorkshire and Meishan testes, although sometimes only by densitometry on Day 30 (Fig. 3). The expression of P450c17 increased dramatically by Day 34 of gestation in both breeds but appeared higher in Meishans than in Yorkshires at this fetal age. The peak levels of expression for Yorkshire fetuses occurred on Day 38 and, though declining slightly, were generally maintained through Day 42 in fetuses of both breeds. The levels of P450c17 protein determined by densitometry of immunoblot autoradiograms were positively correlated with the levels of testosterone secreted by cultured testes from both Meishan and Yorkshire fetuses (r = 0.74, p < 0.09; r = 0.72, p < 0.01, respectively). Additionally, the concentrations of testosterone in culture media were positively correlated with day of gestation (Meishan, r = 0.94, p < 0.01; Yorkshire, r = 0.72, p < 0.01). Testosterone concentrations in medium from cultured testes were generally similar between breeds except on Day 34, when Meishan explants secreted significantly less than Yorkshire testes (2.66 ± 0.95 vs. 7.13 ± 0.76 ng per 6 h, respectively; p < 0.05).



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  		FIG. 3. Western immunoblot analyses of P450c17 expression in Yorkshire (solid bars) and Meishan (open bars) fetal testes recovered from fetuses at Days 30, 34, 38, and 42 of gestation. Means ± SEM were calculated after quantification of each band by scanning densitometry. Bars with different letters differ (p < 0.05) across days within a breed. An asterisk denotes a difference (p < 0.05) between breeds. The inset is a representative immunoblot of Yorkshire (Y) and Meishan (M) fetal testes (25 µg/lane).

In general, testicular cords developed multiple layers of cells and became progressively more defined to Day 42 of gestation in both Yorkshire and Meishan fetuses. The majority of cells within the cords were primitive Sertoli cells. Primordial germ cells were already apparent by Day 30, making a greater contribution to the total cell population of the cords as development proceeded. However, differences were also noted in fetal testicular morphology and development between Meishan (Fig. 4, left panels) and Yorkshire fetuses (Fig. 4, right panels). This was apparent at all stages examined and was associated with differences in staining for PCNA. Specifically, the germinal epithelium of the Meishan testes (Fig. 4A) was characterized by a uniformly arranged, single layer of flattened cells, whereas the cells of the germinal epithelium of the Yorkshire testes (Fig. 4B) were less regular in shape and size. The Yorkshire testes exhibited more obvious cord formation than Meishan fetal testes as early as Day 34 of gestation (compare Fig. 4, C with D). A similar level of compartmental organization was not evident in the Meishan male fetuses until Day 42 (Fig. 4G). Although no morphometric analyses were conducted, the interstitium of the Yorkshire testes appeared generally to be more loosely organized as a result of proportionately more extracellular space than seen in the Meishan testes.



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  		FIG. 4. Immunolocalization of PCNA in sections of porcine fetal testes recovered on Days 30 (A, B), 34 (C, D), 38 (E, F), and 42 (G, H) of gestation from Meishan (left panels) and Yorkshire (right panels) dams. Note the general lack of staining for PCNA in fetal testes of either breed on Day 34. Bar = 100 µm; all micrographs were taken at the same magnification.

Day 30 testes demonstrated positive staining for PCNA in the nuclei of germinal epithelium, the underlying mesenchyme, and, to a lesser degree, the developing interstitium (Fig. 4, A and B). However, the most intense staining for PCNA appeared in the cells of the testicular cords, including both primitive Sertoli cells and primordial germ cells that continued to stain prominently through to Day 42 (Fig. 4, C–H). PCNA staining was particularly strong in the Sertoli and primordial germ cells of the medullary cords formed in Meishan testes on Day 42 of gestation (Fig. 4G). Testes from Days 38 and 42 continued to stain positively for PCNA in both cell types of the testicular cords as well as in cells of the interstitium (Fig. 4, E–H). However, a noticeable decline in staining intensity was observed in testes on Day 34 of gestation (Fig. 4, C and D) in both breeds. Interstitial staining was also consistent on Days 38 and 42. In addition, the number of positive cells and intensity of staining generally were less in the Day 38 Meishan fetal testes than in the Day 38 Yorkshire fetuses.

Measurements of allantoic fluid volume (milliliters) and chorioallantoic (grams) and embryonic weights (grams) were also compared between male Meishan and Yorkshire embryos on Days 30, 34, 38, and 42 of gestation (Table 1). Yorkshire allantoic fluid volume was greatest on Day 30 of gestation and decreased through Day 42. Meishan fluid volume was significantly less on Day 30, increased to levels that were not different from those in Yorkshires by Day 34, and decreased by Day 42 while still remaining greater than those in Yorkshire conceptuses. As was the general case with allantoic fluid volume, chorioallantoic and embryonic weights were greater (p < 0.05) in Yorkshire embryos than in Meishan embryos on Day 30 of gestation; however, by Day 34 of gestation, the effect of breed was no longer evident. Levels of testosterone in allantoic fluid were either below the sensitivity of our assay or found to be not significantly different between breeds (not shown).


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  		TABLE 1. Testes-secreted testosterone, allantoic fluid volume, choriallantoic weight, and fetal weight of Meishan and Yorkshire conceptuses from Days 30, 34, 38, and 42 of gestation.*


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study is the first to demonstrate P450c17 expression in the differentiating fetal Leydig cell, which is evident in the central medullary region and is associated with the initiation of androgen synthesis on Day 30 of gestation. The general morphological development of the porcine fetal testes described herein agrees with that reported by Pelliniemi [21, 22]. Sertoli cells were aligned into discernible cords on Day 30, and the degree of tubular organization continued to become more prominent at each stage. Although the Meishan fetal testes had also developed recognizable testicular cords by 30 days of gestation, tubular development did not parallel that of the Yorkshire and appeared to be delayed by as much as 4 days. The advanced and organized network of testicular cords in the Yorkshire testes at 34 days, and in the Meishan testes at 42 days of gestation, was correlated with regionalized, intense staining for PCNA in cells within the cords.

Immediately following the formation of the testicular cords, which defines general testicular from ovarian morphology (26 days of gestation), fetal Leydig cells become visible within the interstitial regions [21, 22]. Leydig cells increase in size and accumulate large amounts of smooth endoplasmic reticulum, characteristic of steroid-producing cells, from the time they are first observed on Day 27 of gestation [23, 24]. Testicular testosterone concentrations [25], levels of testosterone released during explant culture [26, 27], and serum testosterone concentrations [28] exhibit a pattern similar to that of Leydig cell development. The concentration of testosterone in explant culture media of Yorkshires from the present study agreed with these previous findings. Several of the steroidogenic enzymes necessary for testosterone synthesis are present during the early fetal stage of Leydig cell development [6, 29–31]. The apparently higher level of P450c17 expression in the Meishan than the Yorkshire testes on Day 34 was not associated with greater testosterone secretion and may suggest that other steps are more important in limiting androgen secretion at certain stages of development.

Immunoblot analysis conducted in the present study was able to detect P450c17 expression on Day 30 of gestation, albeit at low levels. Although this was also detectable by ICC and ISH in the center of the medulla of Yorkshire fetal testes, it was not apparent in Meishan testes until Day 34. In situ expression of P450c17 transcript corroborated these findings in both Yorkshire and Meishan testes. This increase coincided with a decrease in the number and intensity of interstitial cells staining for PCNA, suggesting that a decline in interstitial cell proliferation was associated with steroidogenic differentiation of fetal Leydig cells. Moreover, there was consistent, strong PCNA staining in the cells of the testicular cords during this period. The differentiation of mouse gonads, both morphologically and steroidogenically, has been shown to follow the formation and differentiation of testicular cords [31, 32]. This is consistent with observations made in the present study, which demonstrated that the increase in Sertoli cell proliferation and the development of testicular cords roughly parallel the differentiation of the Leydig cells in both Meishan and Yorkshire fetal pigs. These data suggest that the differentiation of fetal Leydig cells, delayed by up to 4 days in Meishan compared to Yorkshire conceptuses, may in turn depend on the differentiation of Sertoli cells.

The synthesis and secretion of Müllerian inhibiting substance (MIS) is a functional marker of Sertoli cell differentiation. Although it is widely accepted that proper duct and external genitalia development is dependent on MIS, it is also possible that this hormone plays a critical role in the development of the testes. MIS activity in the porcine fetal testes increases from negligible levels on Day 27 to a maximum value on Day 33 of gestation [33], which corresponds with the proliferation and organization of the testicular cords in the Yorkshire. Testes of MIS-deficient mice exhibit Leydig cell hyperplasia [34] whereas male transgenic mice overexpressing MIS have feminized external genitalia, have underdeveloped epididymides, and lack seminal vesicles [35]. The absence of wolffian duct development and feminization of the external genitalia in MIS-overexpressing mice could be a result of an androgen deficiency due to MIS-altered Leydig cell differentiation. The delayed development of the Meishan testes may suggest a different pattern of MIS secretion than that observed for the Yorkshire and clearly warrants further investigation.

Information pertaining to the regulation of P450c17 expression in fetal Leydig cells is limited. Although initiation of P450c17 expression in adult Leydig cells depends on LH stimulation [36, 37], no such dependence exists in fetal Leydig cells. Although fetal testicular cells express LH receptors [38] and are modestly responsive to LH [6, 39], the anterior pituitary is not developed at the time of testes differentiation [40, 41]. However, recently discovered transcription factors such as Ad4-binding protein (Ad4-BP) [42] and SRY are known to be expressed immediately before or during the gonadal differentiation in the testicular cords and interstitium of the mouse and rat [43, 44]. Therefore, these and other orphan nuclear receptors may be involved in the ontogenic expression of P450c17 [45]. Research in our laboratory has identified a possible Ad4-BP binding site by sequence analysis in the upstream regulatory region of porcine CYP17 gene encoding P450c17. However, attempts to immunolocalize the protein in the fetal testes using ICC have been unsuccessful (unpublished results). Whether or not this represents differences between species in the transcriptional regulation of P450c17 expression in fetal testes is unclear.

In addition to differences in the rates of embryonic and fetal growth exhibited by the highly prolific Chinese Meishan and domestic Yorkshire breeds of pig [46, 47], significant differences also exist in the reproductive performance of adult animals, including age at puberty, number of sperm per ejaculate, paired testes volume, plasma inhibin and FSH levels, and Leydig cell size [48–50]. The data presented here are consistent with the possibility that these differences may have their origins during fetal development, as early as the time of sexual differentiation. Just how such differences are orchestrated remains to be investigated.


   ACKNOWLEDGMENTS
 
The authors express their thanks to Dr. Diane Keeney for her expert guidance and invaluable help establishing in situ hybridization in our laboratory, and to Dr. S.P. Ford, Carole Hertz, and Matt Wilson for tissue collection and culture.


   FOOTNOTES
 
1 Supported in part by USDA/NRI grants 93-37206-1299 and 94-37203-0717. Back

2 Correspondence. FAX: 530 752 4278; ajconley{at}ucdavis.edu Back

3 Current address: Department of Cell Biology, Neurobiology and Anatomy, College of Medicine, University of Cincinnati, Cincinnati, OH 45267–0521. Back

Accepted: September 1, 1998.

Received: April 21, 1998.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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