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Testis-Like Steroidogenesis in the Ovotestis of the European Mole, Talpa europaea¹

^a Departments of Integrative Biology and ^b Psychology, University of California at Berkeley, Berkeley, California 94720 ^c Department of Zoology, University of Aberdeen, AB24 2TZ, Scotland, United Kingdom

	ABSTRACT

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The female European mole (Talpa europaea) presents a vivid paradox in relation to our contemporary understanding of mammalian sexual differentiation. These animals are exceptional among female mammals in that they possess bilateral ovotestes. The ovotestis contains a morphologically normal ovarian component that develops during the spring breeding season and a histologically defined testicular region, the interstitial gland, which enlarges during autumn when the ovarian component decreases in size. In correlation with this unusual gonadal situation, the female mole displays a penile clitoris traversed by a urethral canal. Although the histology of the ovotestis is well documented and has recently been extended to an additional three species of the genus Talpa, there have been no clear indications of the physiological function, particularly androgen production, of the ovotestis in these female moles. This paper presents the first clear evidence of seasonal variation in plasma testosterone concentrations, which parallel the growth and regression of the "testicular" interstitial gland, in T. europaea. Plasma androstenedione did not show significant seasonal variation, but plasma testosterone (1.06 ± 0.2 ng/ml) and gonadal testosterone concentration (1.57 ± 0.65 µg/mg protein) in females in autumn were significantly higher (p < 0.02) than plasma (0.4 ± 0.2 ng/ml) and gonadal (0.24 ± 0.21 µg/mg) concentrations in pregnant or immediately postpartum females in spring. Our data also reveal selective metabolic production of testosterone from radiolabeled steroid precursors (progesterone and androstenedione) by these ovarian interstitial tissues and male testes; estradiol is produced by ovarian tissue but not interstitial gland or testis.

	INTRODUCTION

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In mammals, sex is determined by the action of the testis-determining gene on the Y chromosome, SRY, which directs the differentiation of the indifferent male gonad into a testis [1–3]. In the absence of a Y chromosome, and SRY, the indifferent gonad follows the default pathway and differentiates as an ovary. The determination of gonadal sex is pivotal to the sexual differentiation of the individual, as subsequent sexual development is mediated by the gonadal hormones [4]. The European mole presents an intriguing contrast to this general mammalian pattern in that all XX females possess ovotestes. There is an annual growth and regression of these tissues, with ovarian follicular tissue predominating during the spring breeding season and testicular-like tissues composing the bulk of the ovary during the autumnal nonbreeding season [5, 6]. The proliferation of testicular-like tissues in the autumn ovary is the inverse of the pattern observed in the male mole, where testicular and androgen-dependent tissues develop in the spring and regress in the autumn [7].

Recently, the presence of ovotestes has been described in an additional three species of the genus Talpa: T. occidentalis, T. romana, and T. stankovici [8, 9]. The ovarian portion of the ovotestis contains morphologically normal follicles, while the adjacent testicular portion, or interstitial gland, consists primarily of well-differentiated Leydig cells with varying numbers of seminiferous tubules [8, 9]. Unlike what is seen in the normal testis, the interstitial gland is devoid of germ cells: all of the germ cells are confined to the ovarian portion of the ovotestis [5, 6, 8, 9]. Early studies describe differentiation of the ovarian and interstitial gland portions of the ovotestis as occurring during fetal development (reviewed in [5]). Females possess a normal vagina, uterus, and oviducts. However, adjacent to the interstitial gland are abnormally developed epididymides, suggesting that the ovotestis secretes testosterone during fetal development [8, 9]. The clitoris is similarly masculinized and develops in the fetus in a remarkably similar fashion to the penis of the male [10] (Fig. 1). Studies in T. occidentalis suggested that plasma testosterone might vary seasonally, but high variability in the data precluded any such conclusions [8].

View larger version (148K): [in this window] [in a new window]   FIG. 1. External genitalia of adult female (left) and adult male (right) Talpa europaea. The female clitoris is traversed by a centrally located urethra. Note the typically larger ano-genital distance of the male. Scale bar represents 5 mm.

The objective of this study was to examine further the hypothesis that the interstitial gland portion of the ovotestis is the primary source of plasma testosterone in the mole T. europaea and to assess the seasonal functionality of these tissues. RIA and in vitro incubation of gonadal tissues were used to examine the capacity of the interstitial gland from adult females to synthesize testosterone and to determine whether testosterone levels vary seasonally, reflecting the change in size of the interstitial gland. In addition, studies of female spotted hyenas, another species with masculinized genitalia, had revealed substantial circulating levels of androstenedione, primarily of ovarian origin, throughout life [11], and exceptional elevation of testosterone concentrations during pregnancy [12]. The latter was found to result from placental conversion of androstenedione to testosterone and was tentatively linked with genital masculinization of the developing fetus [13]. We accordingly assessed plasma androstenedione concentrations in female moles in order to determine whether a similar tendency toward generally elevated androstenedione levels, or pregnancy-elevated testosterone levels, are associated with masculinization of the genitalia in Talpa.

	MATERIALS AND METHODS

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Animals

Twenty-three animals were trapped live in the vicinity of Newcastle, England, in late April–early May and mid-November, corresponding to the Northern Hemisphere spring and autumn, respectively. Animals were returned to the laboratory in Aberdeen (approximately 4 h), where they were weighed, killed, and dissected. Ovotestes and testes were frozen on dry ice before storage at -80°C. Plasma was separated from whole blood by centrifugation and stored at -80°C.

In Vitro Incubations

Ovotestes from spring females were left intact, whereas those from autumn animals were more easily dissected into their respective ovarian and interstitial gland portions; however, it should be noted that while we were successful in removing ovarian tissue from the interstitial gland, the ovarian portion invariably contained some contaminating interstitial gland. Frozen gonads were homogenized individually in 500 µl of 0.05 M Tris (pH 7.4) with a glass microhomogenizer. The objective of these studies was to determine the ratio of androgens to estrogens in ovotestes of reproductively active and inactive females. Consequently, protein content was not quantified for each sample; rather, gonadal homogenates were diluted such that after the incubation period not all of the precursor steroid was metabolized. Homogenates were brought to a final volume of 3 ml with 0.05 M Tris. The following cofactors were added: NADP at 1 mM, glucose-6-phosphate at 10 mM, and glucose-6-phosphate dehydrogenase at 5 U/ml (Sigma Chemical Co., St. Louis, MO). [4-¹⁴C]Progesterone (0.1 µCi; NEN, Boston, MA) or 1 µCi of [1,2,6,7-³H]androstenedione was added to the homogenates. Samples were incubated at 37°C with continuous agitation for 30 min with [¹⁴C]progesterone or for 15 min with [³H]androstenedione: these incubation times ensured that the entire radiolabeled steroid was not metabolized by the end of the incubation period. Steroids were extracted twice with ethyl acetate (Fisher, Santa Clara, CA). The remaining aqueous phase was counted in a scintillation counter to determine the percentage recovery of labeled steroids, which was greater than 95%. Ethyl acetate was evaporated from the extracted steroids with nitrogen at 45°C before resuspension in 50 µl of ethyl acetate. Steroid metabolites in each sample were identified by thin-layer chromatography.

Thin-Layer Chromatography

Samples were loaded onto Whatman (Clifton, NJ) 20-cm x 20-cm Linear K TLC plates with silica gel as absorbent (Fisher). Each sample was loaded onto two plates; one was developed in chloroform:methanol (98:2), the other in benzene:ethyl acetate (3:1). Standard steroids were [³H]progesterone, androstenedione, testosterone, dihydrotestosterone, and estradiol (NEN). Radioactive metabolites were detected by a Berthold (Wildbad, Germany) TLC scanner. To confirm the identity of the metabolites, regions corresponding to the standards were scraped from the plate, reextracted with ethyl acetate, and run on a new plate in the solvent that was alternative to the original.

RIA

The concentration of testosterone in plasma and gonadal homogenates was determined by RIA as described in detail by Licht et al. [14]. Briefly, plasma samples and gonadal homogenates were extracted twice with diethyl ether (Mallinkrodt, St. Louis, MO), and testosterone was measured with an antiserum cospecific for testosterone and dihydrotestosterone. [1,2,6,7-³H]Testosterone was used as the label (NEN). Measurements of plasma androstenedione were determined by RIA as described by Glickman et al. [11]. Plasma samples were extracted as above and assayed with an antiserum specific for androstenedione with [1,2,6,7-³H]androstenedione as label (NEN).

Protein Assay

Protein content for each sample was determined by Bradford protein assay: 160 µl of serial dilutions of the gonadal homogenates was added to a microtiter tray with 40 µl of protein assay dye reagent (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Serial dilutions of BSA were used as the standards. Absorbance was determined by a Bio-Rad UV microplate reader.

Statistics

Significance between groups was assessed with the nonparametric Mann-Whitney U Test and are presented as means ± SD.

	RESULTS

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Gonadal Size and Testosterone Concentrations in Plasma and Gonads

In autumn, when the animals are reproductively inactive, the ovarian portion exists as a small cap of tissue. At this time, in contrast, the interstitial gland reaches its maximum size, which is significantly greater (p < 0.002), by about 2-fold, than during the spring period of reproductive activity—when the ovarian portion increases in size as follicles mature and the interstitial gland decreases in size (Table 1). Body weight does not differ significantly between the two seasons (data not shown).

Females in autumn had a significantly higher concentration of circulating testosterone than pregnant females in spring (p < 0.01, Table 1). Plasma androstenedione did not differ significantly (p > 0.2) between these two groups. These levels of testosterone in autumn approached those of the two males collected in this season (Table 1). Interestingly, the one nonpregnant female in the spring had one of the highest plasma testosterone concentrations of all of the females examined (Table 1; Fig. 2). Among the reproductive females, there were no differences in plasma testosterone between the 3 that were immediately postpartum (within 24 h of parturition) and the 11 pregnant females, and plasma testosterone did not correlate with stage of pregnancy (as judged by the size of the fetoplacental units). Due to small sample size and the exceptional variability observed in our male moles in the spring, no statistical tests were conducted. However, in keeping with prior observations [7], there was a tendency for spring males to display higher concentrations of testosterone than males sampled in the autumn.

View larger version (17K): [in this window] [in a new window]   FIG. 2. Relationship between plasma testosterone and total gonadal testosterone in male and female moles during spring and autumn. Note that plasma testosterone, but not total testosterone, was available for two pregnant females in spring (< 0.1 and 0.41 ng/ml), one female in autumn (0.8 ng/ml), and one male in autumn (1.5 ng/ml). Consequently, these data have been omitted from this figure.

To determine whether the elevated levels of circulating testosterone observed in autumn females were attributable simply to the growth of the interstitial gland, or were also the result of an increase in the ability of the Leydig cells to synthesize testosterone, we assayed testosterone concentration in gonadal homogenates. Ovotestes from reproductively inactive females contained significantly more testosterone per milligram of protein than ovotestes from pregnant females in spring (p < 0.01, Table 1). Although the ovarian portion of ovotestes collected in spring is larger than that of ovotestes collected in autumn, the increase in ovarian tissue was not sufficient, relative to the size of the interstitial gland, to account for the lesser testosterone content per milligram of protein observed for these ovotestes. The one nonpregnant female collected in spring produced levels of testosterone that, per milligram of protein, were comparable to those of reproductively inactive females—reflecting the high levels of circulating testosterone observed in this animal (Table 1). The size of the ovotestes from this one nonpregnant female in the spring was within the range for those collected from the pregnant animals, but the structure was distinctive. In the nonpregnant individual the yellowish interstitial gland was distinct from the white ovary (follicular), whereas no such distinction could be made for the ovotestes of the 14 reproductively active (pregnant or postpartum) females.

The testes of one of the two spring males examined contained more testosterone per milligram of gonadal protein than did those of males collected in autumn (Table 1). However, the testosterone concentration in the testes of the other spring male was considerably lower (Table 1).

When testosterone content per milligram of gonadal protein was corrected to testosterone content per total gonadal weight in milligrams, the increase in testosterone content for autumn ovotestis compared to spring ovotestis was highly significant (p < 0.001, Table 1). When individual testosterone contents per total gonadal weight were plotted against plasma testosterone, the animals fell into three distinct groups: spring pregnant females with the lowest values, autumn females and the one spring nonpregnant female (i.e., nonreproductive females) with significantly higher values (p < 0.001), and males with the highest values (Fig. 2).

Production of Testosterone In Vitro

Interstitial glands from reproductively inactive females metabolized the [¹⁴C]progesterone into predominantly testosterone at levels comparable to those observed for the testes from reproductively inactive and active males (Table 2). In contrast, the ovarian portion of the ovotestis from reproductively inactive females converted significantly less of the [¹⁴C]progesterone into testosterone (p < 0.029, Table 2) and, unlike the interstitial gland, also synthesized estradiol (Table 2). It is likely that contaminating interstitial gland contributed to the production of testosterone in these samples. We were unable to separate the ovotestes from one female into their respective interstitial gland and ovarian portions, and so they were incubated intact. As expected, testosterone and estradiol were both synthesized at levels similar to those observed for the animals from which the respective portions had been incubated separately (Table 2). Intact ovotestes from pregnant females in spring did not produce detectable amounts of testosterone but did synthesize estradiol (Table 2). Similarly, ovotestes from the one nonpregnant female captured in spring metabolized the [¹⁴C]progesterone into estradiol (Table 2).

Similar relative trends were observed when tissue homogenates were incubated with [³H]androstenedione. Interstitial glands from reproductively inactive females metabolized the [³H]androstenedione into predominantly testosterone (Table 3). The ovarian portion from these animals produced significantly less testosterone than the interstitial gland (p < 0.05), and it is possible that contaminating interstitial gland contributed to the testosterone produced. In addition, the ovarian portion synthesized estradiol and an unidentified ovary-specific metabolite (Table 3). Intact ovotestes from the one reproductively inactive female produced testosterone, estradiol, and an ovary-specific metabolite at levels comparable to those observed for the respective portions when incubated separately (Table 3). Intact ovotestes from pregnant females in spring produced significantly less testosterone than the interstitial gland from reproductively inactive females (p < 0.001, Table 3). Similar results were observed for the one nonpregnant female collected in spring (Table 3). Testes from reproductively active and inactive males synthesized only testosterone (Table 3).

	DISCUSSION

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The testis-like histology of the interstitial gland is reflected in its testis-like steroidogenic activity. Circulating levels of testosterone in the reproductively inactive females were exceptionally high for any nonpregnant female mammal: T. europaea, 0.79–1.44 ng/ml; spotted hyena, 0.40–0.50 ng/ml [11]; rhesus monkey, 0.20–0.80 ng/ml [15]; Siberian tiger, 0.20–0.70 ng/ml [16]; rat, 0.16–0.18 ng/ml [17]; rabbit, 0.05–0.10 ng/ml [15]; dog, 0.030–0.7 ng/ml [18, 19]; cow, 0.01–0.02 ng/ml [20]. In contrast, during the period of reproductive activity, levels of circulating testosterone returned to within typical female levels of 0.24–0.68 ng/ml. Jimenez et al. [8] found a similar trend in seasonal testosterone levels in T. occidentalis; however, because of high variation between individuals, no clear relationship between reproductively active and inactive animals could be defined. The substantial circulating levels of androstenedione observed during both spring and autumn indicate that this steroid may provide a substrate for the metabolic production of testosterone as was observed in vitro.

The ovotestes of reproductively inactive females contained, per milligram of gonadal protein, around eight times as much testosterone as the ovotestes from pregnant females, while the increase in ovotestis weight was only around 2-fold. Consequently, the increase in plasma testosterone in reproductively inactive females likely results from both the growth of the interstitial gland and an increased synthesis and secretion of testosterone by the Leydig cells of the interstitial gland. Early histological studies support these data. In a detailed study of the histology of the interstitial gland, Deanesly [6] indicates that the interstitial cells of the anestrous ovotestis are large and glandular and that they contain lipoids, while those from pregnant females are regressing and vacuolated. Also noteworthy in this regard is the similarity of plasma testosterone levels in autumn females and males, despite an order of magnitude higher concentration and content of gonadal testosterone in males (Table 1).

Perhaps the most striking aspect of the ovotestis is the cyclical nature of steroidogenic activity between the ovarian and interstitial gland components. This suggests that steroidogeneses in the ovary and interstitial gland are under independent control—a situation at odds with our current understanding of the regulation of gonadal steroidogenesis. Ordinarily, gonadotropins signal to the steroidogenic cells of the gonad to increase the synthesis of a particular steroid. In T. europaea, the interstitial gland is up-regulated in its production of testosterone while the ovarian portion is in its least active phase of steroidogenesis. Similarly, when ovarian production of androstenedione and estradiol is up-regulated during estrus, the secretion of testosterone by the interstitial gland is substantially diminished. It seems paradoxical that steroidogenesis is up-regulated in only one portion of the gonad at any given time. It is possible that the receptors for LH and FSH may be differentially expressed in the ovarian and interstitial gland portions. Alternatively, another hormone, distinct from the gonadotropins, may differentially affect steroidogenesis in the ovary and interstitial gland. Understanding the mechanism by which this unique situation is permitted to occur will further our understanding of the regulation of gonadal steroidogenesis and reproductive function.

The phallic-like clitoris of the female mole prompts comparisons with that of the female spotted hyena (Crocuta crocuta), where the clitoris is similarly traversed by the urethra [5, 21, 22]. However, in contrast to what is seen in the spotted hyena, where a striking elevation of maternal androgens during pregnancy is thought to contribute to the masculinization of the female fetus [12, 13], these studies did not show elevated levels of maternal androgens during pregnancy. Thus, masculinization of the mole clitoris may be directed by the production of androgens by the fetal interstitial glands that are not reflected in elevated androgens in the maternal circulation.

T. europaea is one of four species of the genus Talpa known to have ovotestes. Of interest is how this phenotype could have arisen in the ancestor of the genus Talpa and what advantage this phenotype conveyed in order for it to be conserved in every female of all four of the species examined. Because the basic form of the ovotestis is found in at least four species of mole, Sanchez et al. [9] have argued that it is an evolutionarily primitive condition resulting from an ancient mutation. It seems likely that female moles have evaded the common costs of exceptional androgen production on reproductive success [23] by the reciprocal, seasonal inversion of the two component tissues of the gonad; persistently high levels of testosterone during spring would be expected to be disruptive to folliculogenesis and estrus. However, these considerations leave open the question of what benefits might accrue from higher testosterone production in autumn. In a comprehensive review of the natural history of moles, Gorman and Stone [24] write, "there is no doubt that the medullary (i.e., interstitial) tissue is endocrine in nature and involved in the synthesis of hormones, but what hormones and to what purpose is as yet unknown" (pp. 60–61). The authors then add, "One possibility is that for most of the year the medullary (interstitial) tissue produces male hormones and that these are responsible for the aggressive nature of the female and for the masculine appearance of her genitalia." Data presented in the current paper clearly establish that testosterone is a primary metabolic product of the medullary/interstitial tissues resulting in elevated plasma concentrations of this steroid. In accordance with the preceding scenario, Gorman and Stone [24] note that, unlike other female insectivores (e.g., shrews), female T. europaea do not increase the size of their territories during the breeding season (pp. 93–94). These moles appear to maintain a sufficiently large territory year-round to deal with the increased energetic demands of lactation. Both sexes expend significant energy in digging tunnels that form pitfall traps for earthworms, the major component of their diet. Consequently, individuals defend their tunnels and food supply aggressively [24]. However, this aggression must be reduced in females during the short period of estrus to allow mating. Further research will be required to determine whether female moles with high testosterone concentrations are more successful in aggressive encounters when establishing/maintaining such territories outside of the breeding season, and whether the androgens play a role in the formation of the penile clitoris.

	ACKNOWLEDGMENTS

 
We are indebted to Dr. Lawrence G. Frank, who arranged the collaboration between Aberdeen and Berkeley that led to the present manuscript and to Dr. Jean Wilson, who encouraged us to pursue the problems presented by female European moles. Dr. Tyrone Hayes and Dr. Jennifer Hugenberger provided assistance with the thin-layer chromatography and Bradford protein assays, respectively. Androstenedione antiserum was generously provided by Dr. Scott Monroe. We thank Dr. Alisdair Daws for critically reading the manuscript. The research was supported by a grant from the National Institute of Mental Health (MH-39917).

	FOOTNOTES

 
¹ Supported by grant MH-39917 from the National Institute of Mental Health.

² Correspondence: Deanne J. Whitworth, Department of Molecular Genetics Box 45, University of Texas, MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. FAX: 713 794 4394; deanne_whitworth{at}molgen.mdacc.tmc.edu

Accepted: September 21, 1998.

Received: June 12, 1998.

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