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RESEARCH |
University of Cambridge, Department of Veterinary Medicine Equine Fertility Unit, Mertoun Paddocks, Woodditton Road, Newmarket, Suffolk CB8 9BH, UK and 1 University of Pretoria, Mammal Research Unit, Pretoria 0002, South Africa
Correspondence should be addressed to W R Allen; Email: efu{at}tesco.net
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Abstract |
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-dihydroprogesterone and other 5-reduced progestagens from cholesterol and pregnenelone and the hypothesis is raised that these fetal gonadal progestagens may supplement significantly the progestagens secreted by the multiple large corpora lutea of pregnancy in the elephant. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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-hydroxylated C19 steroids (Pashen & Allen 1979, Tait et al. 1983, 1985), which the placenta then aromatizes to the common phenolic oestrogens, oestrone and oestradiol, and the equine-specific ring ß unsaturated oestrogens, equilin and equilenin (Bhavnani et al. 1969, 1971, Bhavnani & Short 1973a, 1973b). These oestrogens are present in high concentrations in the blood and urine of pregnant equids between 100 and 320 days of gestation (Cox 1975, Raeside & Liptrap 1975).
The gonads of the elephant fetus undergo a similar interstitial cell-driven enlargement during the second half of gestation (Perry 1964, Hanks 1971) and these cells have been shown recently to possess the steroid synthetic enzymes, P450 side-chain-cleavage enzyme (SCC 450) required for the conversion of cholesterol to pregnenelone, and 3ß-hydroxysteroid dehydrogenase (3ßHSD) involved in the metabolism of pregnenelone to progesterone and other progestagens (Allen et al. 2002). Furthermore, elephant fetal gonad tissue recovered after the tenth month of gestation metabolized tritium-labelled cholesterol and pregnenelone to 5-dihydroprogesterone and other 5-pregnane derivatives in vitro (Allen et al. 2002).
An opportunity was afforded to examine the phenomenon of gonadal enlargement in the elephant fetus more closely in 44 fetuses recovered from pregnant elephant culled for management reasons in South Africa. This paper describes the morphological changes in these fetal ovaries and testes throughout gestation and discusses the possible role(s) of the fetal gonads in the maintenance of pregnancy in this species.
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Materials and Methods |
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Histological preparation of tissues
Small blocks (approximately 2 cm3) of tissue cut from one of each pair of gonads were fixed in Bouin’s fluid for histological examinations, snap-frozen in OCT Embedding Compound (Raymond Lamb, East Sussex, UK) in liquid nitrogen for immunocytochemical staining or immersed in cold PBS for steroid hormone synthesis and conversion experiments (Allen et al. 2002). The Bouin’s fixed samples were dehydrated by passing them through increasing concentrations of alcohol followed by xylene. They were embedded in paraffin wax and sectioned at 5–8 µm for staining with haematoxylin and eosin.
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Results |
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, Fig. 1). Similarly, 21 pairs of testes with a mean individual weight of 0.063–22.5 g were recovered from male fetuses weighing 0.09–112 kg (6.1–21.3months’ gestation; Table 1b, Fig. 1).
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), whereas the testes remained essentially spherical in shape throughout pregnancy (Fig. 3a). The gonads of both genders increased markedly in size from around the eleventh or twelth month of gestation, with the growth spurt being more pronounced in the ovaries (e.g. 2.2 rising to 14.1 g between 11.3 and 13.5 months’ gestation; Table 1a
) than the testes (e.g. 2.1 rising to 5.8 g between 12.3 and 13.7 months’ gestation; Table 1b). Thereafter, the ovaries grew rapidly to reach a maximum mean weight of 30–35 g at around 16–17 months (Table 1a, Fig. 1) compared with a maximum mean testis weight of only 20–23 g, again around 16–17 months (Table 1b, Fig. 1). Beyond this stage of maximum growth, the ovaries showed a much more pronounced decline in size than the testes until the point was reached when the weights of the gonads of both genders levelled off and fluctuated around 15–23 g during the last 2–3 months of gestation (Fig. 1).
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), the ovaries developed a markedly cobbled external appearance in mid-gestation due to the enlargement and antrum formation in many follicles in both the medullary and cortical regions of the ovary (Fig. 2a and b). This external roughness tended to smooth out and disappear in the final stages of gestation, however, in parallel with a decline in the number and size of antral follicles (Fig. 2f). It was evident that the faster and more pronounced increase in weight of the ovaries over the testes in mid-gestation, followed by the more rapid decline in the former, was caused by the growth and fluid accumulation in the antral follicles in the ovaries followed by their regression and atresia (Table 1, Figs 1, 2a and 3a).
Histological findings
Fetal ovaries
At the earliest stage examined histologically (cull no. E70/95, 650 g fetus, 7.6 months’ gestation; Table 1a) the overall picture was much as one would expect to see in the ovaries of a late-stage fetus or newborn offspring of any large domestic species. Namely, a dense accumulation of large, lightly stained oocytes (Fig. 2c), most of which were surrounded by a single layer of rather flattened follicle or putative granulosa cells, to create primitive or primordial follicles in the cortical region of the ovary. Cords of these primordial follicles extended through the dense, fibrotic ovarian stroma towards the central, medullary region of the organ (Fig. 2c) and there was no sign of any follicular enlargement or interstitial cell development at this early stage.
The next stage examined (cull no. E42/94, 7.4 kg, 11.3 months; Table 1a) showed some marked changes. Most notably, considerable enlargement and antral formation in many of the follicles throughout the cortex and medullary regions of the ovary, interspersed with discrete zones of mononucleate interstitial cells (Fig. 2d), which appeared to be originating by hypertrophy and rounding up of the fibroblast-like stromal cells (Fig. 2e). This enlargement caused the interstitial cells to cluster tightly together in distinct groups or zones between the enlarging follicles (Fig. 2d). In this manner the interstitial cell zones and the antral follicles quickly came to constitute the bulk of the ovarian tissue, leaving only sparse trabeculae of normal fibrous stroma scattered throughout the organ.
As gestation advanced to 10–18 months, in which the fetal ovaries showed a more marked increase in growth compared with the fetal testes (cull no. E74/95, 17 kg, 13.5 months; Table 1a), the enlarged antral follicles appeared both bigger and more numerous within the ovarian stroma and the zones of interstitial cells were now much broader due to increased recruitment and hypertrophy of the interstitial cells. Most of the antral follicles exhibited a thin wall (Fig. 2d) composed of a single layer of primitive fibroblast-like cells but large oocytes were occasionally seen within the follicles, each enveloped in a typically multi-layered cumulus and usually attached to a more multi-layered follicle wall.
Continuing through gestation towards term, the number and size of enlarged antral follicles declined steadily from around 18 months of gestation (Fig. 2f; cull no. E51/94, 98.5 kg, 19.9 months; Table 1a) and patches of lymphocyte-like mononuclear cells could be seen infiltrating into the spaces occupied by the previously enlarged antral follicles (Fig. 2g; 98.5 kg). The interstitial tissue was still the most prominent component of the ovarian stroma but, in this latest stage ovary examined (cull no. E51/94, 98.5 kg), interstitial cells at the periphery of the zones could be clearly discerned to be degenerating.
Fetal testes
At the earliest stage examined histologically (cull no. E34/95, 90 g fetus, 6.1 months’ gestation; Table 1b) the fetal testes already exhibited an organized arrangement of small lobular accumulations of interstitial cells in which sparsely arranged primitive seminiferous tubules were embedded, with the lobes separated by loosely arranged fibrous trabeculae containing the blood vessels. As gestation advanced, the whole testis enlarged steadily (see Table 1b) as a result of an increase in the bulk of the individual lobes of interstitial cells. This caused the lobes to become more tightly packed together (Fig. 3b; cull no. E38/94, 18.8 months; Table 1b) with a consequential reduction in the width of the separating fibrous trabeculae. As in the fetal ovaries, it was evident that the interstitial cells originated from phenotypic transformation and hypertrophy of the fibroblast-like stromal cells (Fig. 3c; cull no. E38/94).
The lobular accumulations of interstitial cells persisted until the latest stage of gestation examined (cull no. E33/95, 99 kg, 20.7 months; Table 1b). However, they reduced slowly in size (Fig. 3d; cull no. E33/95) and the seminiferous tubules within each lobe appeared to both multiply in number and become more tightly coiled, thereby increasing their density within the interstitial cell parenchyma (Fig. 3e; cull no. E33/95). As in the fetal ovaries, clusters of interstitial cells at the periphery of the lobes could be seen to be degenerating but, in contrast to the ovaries, this process did not appear to attract infiltrations of mononuclear cells.
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Discussion |
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Other differences between the fetal gonads of horses and elephants include the steroidogenic capacities of their interstitial cell components. In equids, the cells show a remarkable capacity to synthesize a range of C19 androgens from cholesterol, and from precursor molecules even earlier in the steroid synthetic pathway, such as squalene and mevalonate (Bhavnani & Short 1973a, 1973b). These include androstenedione and dehydroepiandrosterone, which are rapidly aromatized to oestrone and oestradiol by the aromatase-containing placental tissue (Bhavani et al. 1969) and also unusual 7-hydroxylated forms of both these androgens which the placenta aromatizes to the equine-specific ring ß unsaturated oestrogens, equilin and equilenin (Tait et al. 1983, 1985). In this way the equine fetal gonads perform a pivotal role in producing the very high concentrations of both phenolic and ring ß unsaturated oestrogens that are present in the blood and urine of the mare during the second half of pregnancy (Cox 1975, Raeside & Liptrap 1975) and which appear to be important in promoting fetal growth and maturation and in preparing the myometrium and other uterine tissues for parturition (Pashen & Allen 1979).
The persistence of sizeable accumulations of apparently healthy interstitial cells in both the testes and ovaries of the elephant fetus right up to term may reflect a biological need for progestagens synthesized by the fetal gonads (Allen et al. 2002) to be added to those coming from the two to eight large plum-like corpora lutea which have been observed to persist in the ovaries of the pregnant elephant throughout gestation (Laws 1969, Hodges 1998, Allen et al. 2003). In a recent elegant study, Meyer et al.(2004) measured the profiles of progestagens, prolactin, relaxin and cortisol in serial peripheral vein blood samples recovered throughout pregnancy from 19 Asian and 8 African elephants maintained in zoos across America. Individual progestagen profiles in both species showed a pronounced fall and secondary rise around the end of the first month of gestation, similar to that seen in the pregnant mare around days 35–40 after ovulation in conjunction with the occurrence of the first of what becomes a series of secondary ovulations stimulated by the luteinizing hormone-like activity of equine chorionic gonadotrophin secreted by the fetal endometrial cups (Allen 1975, Squires & Ginther 1975, Daels et al. 1991). Thereafter in the pregnant elephant, progestagen levels rose steadily to a peak around the fifth month of gestation, remained relatively constant for the next 5–6 months and then, at least in the African elephant, declined quite sharply again to a lower plateau which was then maintained until a sudden final drop 1–2 days before birth. It is interesting to speculate that this late gestation decline in levels may mirror some sort of degeneration and reduction in secretory activity of the ovarian corpora lutea and the replacement of the missing progestagens by those secreted from the fetal gonads in the last months of gestation. Such an hypothesis would be supported by the original observations made by Laws (1969). When serially sectioning the ovaries of 109 pregnant elephants culled in Uganda and Kenya, he noted a definite decline in the total weight of luteal tissue in both ovaries during the second half of gestation.
Meyer et al.(2004) confirmed the earlier findings of McNeilly et al.(1983) that a pronounced increase in serum prolactin concentrations occurs in pregnant elephants from around the fifth month of gestation. They also made the intriguing observation of significantly higher mean progestagen concentrations in the blood of Indian, but not African, elephants carrying male as compared with female fetuses. Perhaps this reflected a lower contribution of progestagens from the fetal ovaries in the later stages of gestation due to the enlarged follicles restricting the total volume of progestagen-secreting interstitial cells that could develop. But why the same situation should not pertain in the African elephant fetus, in which the present study has confirmed the enlargement of multiple ovarian follicles, remains a puzzle.
Another mystery in the pregnant elephant involves the nature and source of the gonadotrophic and/or luteotrophic stimuli for both the development and persistence of the multiple large corpora lutea of pregnancy and the enlargement and steroidogenic function of the fetal gonads. Hodges (1998) questioned whether the elephant is monovular or polyovular and, after discounting the earlier hypothesis of Short (1966) that the multiple corpora lutea encountered in pregnant and non-pregnant elephants might reflect accumulation of luteal structures from cycle to cycle in order to achieve a critical mass of productive luteal tissue to support pregnancy, he concluded that the formation of multiple corpora lutea, with and without ovulation stigmata, probably occurs in each oestrous cycle, with structural – but not functional – persistence into subsequent cycles. Hodges and his colleagues also confirmed the original findings of Short & Buss (1965) and Smith et al.(1969) of the complete absence of native progesterone in the luteal tissue and peripheral blood of cycling and pregnant African elephants and its replacement by 5-reduced progestagens, especially 5-dihydroprogesterone (Heistermann et al. 1997a, 1997b, Hodges et al. 1997). Coincidentally, Meyer et al.(1997) and Greyling et al.(1998) determined that 5-dihydroprogesterone exhibits higher affinity for the endometrial progesterone receptor in the elephant than progesterone itself. Taken together, these two findings highlight the likelihood that, as has been demonstrated in the mare during the second half of gestation (Hamon et al. 1991, Holtan et al. 1991), 5-dihydroprogesterone, rather than progesterone, is the most biologically significant progestagen in the elephant in terms of support for the pregnancy state.
With regard to gonadotrophic and/or luteotrophic stimulation in pregnancy, McNeilly et al.(1983) were unable to detect any differences in serum levels of immunoreactive follicle-stimulating hormone and luteinizing hormone between cycling and pregnant elephants, and Allen et al.(2002) could find no hint of biological or immunological gonadotrophic activity in five saline extracts of placental tissue recovered from pregnant elephants between 4 and 11 months of gestation. This was perhaps not surprising in view of the zonary endotheliochorial nature of the elephant placenta and the complete absence of binucleate cells or syncytial formation in the trophoblast layer (Wooding et al. 2005). Furthermore, as discussed by Maston & Ruvolo (2002), the secretion of gonadotrophic hormones by the placenta is relatively recent in evolutionary terms, even among the primates, and equids are the only other non-primate genus known to have developed this placental function. The pronounced rise in serum prolactin levels after 5 months of gestation in the elephant (McNeilly et al. 1983, Meyer et al. 2004) offers the possibility that prolactin, most likely of maternal pituitary rather than placental origin, may give essential luteotrophic support to the multiple large corpora lutea of pregnancy. However, it is difficult to imagine that prolactin could also provide the gonadotrophic stimulus necessary to induce interstitial gland cell development, follicular growth and steroidogenesis in the fetal gonads. This being so, and in the absence of a placental gonadotrophin, premature activity of the fetal pituitary gland seems the last remaining option and the most likely source of this mid-gestation gonadotrophic drive. Clearly, much more research is needed to provide answers to these and many other fascinating questions concerning the controlling mechanisms and biological roles of gonadal enlargement in the fetal elephant.
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Acknowledgements |
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Footnotes |
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(Present address of M Ford: 18 Belmonte, 8 Maiana Avenue, Brummeria, Pretoria 0184, South Africa)
Received 17 February 2005
First decision 28 April 2005
Revised manuscript received 9 June 2005
Accepted 4 August 2005
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References |
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