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ABSTRACT

Iron is required for the normal development of germ cells during spermatogenesis. Because these cells have no direct access to systemic iron, there exists a shuttle system involving production and secretion of the iron-transporting protein transferrin by the Sertoli cells. Previous reports using cultures of immature Sertoli cells exposed to adult germ cells, or in vivo studies involving germ cell-depleted adult rat testes, concluded that production of transferrin by Sertoli cells is modulated by germ cell complement.

In the present study we have used in situ hybridisation with cRNA probes directed against the 5′ and 3′ ends of transferrin mRNA to examine the pattern of expression of transferrin in the immature and adult rat testis. Adult rats were treated with ethane dimethane sulphonate or methoxyacetic acid (MAA) to manipulate their testosterone levels or germ cell complement respectively. Initial findings obtained using the 3′ probe showed a decrease in transferrin mRNA associated with round spermatid depletion. However, these data were not confirmed by in situ hybridisation when the 5′ probe was used. The specificity of the probes was examined using Northern blotting and the 3′ probe was found to hybridise to the germ cell transcript for hemiferrin even under conditions of high stringency. Examination of immature and pubertal rat testes by in situ hybridisation using the 5′ transferrin-specific probe found that as early as 14 days of age the level of expression of transferrin mRNA was clearly different between tubules, and the mRNA appeared to be expressed in Leydig cells on and after day 31. In the adult rat testis, maximal expression of transferrin mRNA was found at stages VIII-XIV, calling into question the interpretation of the results of some previous studies showing expression of transferrin mRNA at all stages of the spermatogenic cycle. This stage-specific pattern of expression was not altered by acute germ cell depletion using MAA. However, Northern blot analysis showed a statistically significant increase in transferrin mRNA expression at 7 days after MAA treatment when pachytene spermatocytes were depleted from tubules at all stages of the spermatogenic cycle at which transferrin is normally expressed.

In conclusion, we found that transferrin mRNA expression was not modulated by round spermatids as has been reported previously but that meiotic germ cells may influence expression of transferrin at specific stages of the spermatogenic cycle.

ABSTRACT

Oestrogen receptor mRNA expression in mouse ovaries was analysed by Northern blotting of total RNA using ³²P-labelled RNA probes complementary to different functional domains of the oestrogen receptor. The ~6·5 kb mouse oestrogen receptor mRNA transcript was present in immature and adult ovaries at extremely low abundance compared with uterus and oviduct. Using a probe complementary to the steroid-binding domain of the oestrogen receptor (probe EF), a novel RNA transcript of ~1·5 kb was also found in the ovaries but was absent from uterus and oviduct. The melting temperature of the hybrid produced by the ~1·5 kb transcript with probe EF was ~10°C lower than that produced by authentic oestrogen receptor mRNA, which demonstrates incomplete sequence homology between the two transcripts and indicates that the ~1·5 kb RNA is not a truncated form of oestrogen receptor mRNA. Furthermore, the ~1·5 kb RNA lacks the DNA-binding domain found in the oestrogen receptor. The ~1·5 kb RNA, but not oestrogen receptor mRNA, was enriched in total RNA from isolated granulosa cells compared with residual ovarian tissue. The encoded product of this novel oestrogen receptor-related RNA could be a steroid-binding protein involved in oestrogen action in the ovaries.

ABSTRACT

Localization of inhibin/activin subunit mRNAs within the macaque ovary from the immediate pre-ovulatory period of the menstrual cycle, when serum immunoreactive inhibin begins to rise, to day 9 of the luteal phase, when serum inhibin concentrations are maximal, was investigated using in-situ hybridization. Ovaries were studied on the day of the LH surge (day 0) and on days 2, 5, and 9 of the luteal phase by hybridizing frozen tissue sections with radio-labelled riboprobes specific to the inhibin/activin α-, β_A-and β_B-subunits. After autoradiographic exposure for 10 and 21 days, grain concentrations were quantified by image analysis. Moderate expression of α-, β_A- and β_B-subunit mRNA was present within the granulosa cells of the pre-ovulatory follicle (day 0). The granulosa-lutein cells of the corpora lutea expressed high levels of α-subunit at days 2, 5 and 9. mRNAs for β_A and β_B were detected at low but significant levels in all of the corpora lutea. All healthy antral follicles exhibited a high level of expression of β_B-subunit mRNA in the granulosa cells. On day 2 after ovulation these follicles also expressed high α- and moderate β_A-subunit mRNA. On day 9 the β_B-inhibin mRNA in antral follicles was found in association with low expression of the other subunits. Small follicles in ovaries on day 2 expressed moderate α- and low levels of β_B-subunit mRNA, while mRNA for β_A was absent. α-subunit mRNA expression was present on day 5 while neither β_A- nor β_B-subunit mRNA was detected. On day 9 a proportion of small follicles expressed α- and β_A-subunit mRNA. These results demonstrate that marked differences are present in the levels of expression of the three inhibin/activin subunit genes between follicles and the corpus luteum. The predominance of the β_B-subunit mRNA within antral follicles would be consistent with the synthesis of activin. The predominance of the α-subunit combined with the low expression of the β-subunits in the corpus luteum suggests that both biologically active inhibin and free α-subunit are produced by the primate corpus luteum.

ABSTRACT

While the fetal pituitary synthesizes and releases prolactin, it is also produced within the utero-placental unit during pregnancy in women and has been localized in the amnion, chorion and decidua. However, it is not clear whether prolactin is synthesized within all these non-fetal pituitary tissues. We have investigated prolactin production and its gene expression using tissue culture, immunocytochemistry and in-situ hybridization techniques. Prolactin was immunolocalized not only in the decidua but also in amnion and trophoblast cells. In contrast, the in-situ hybridization results showed that silver grains, formed by specific hybridization of a prolactin cDNA probe to prolactin mRNA, were confined to decidual cells of early and term pregnancy. The results from tissue cultures correlated well with those of in-situ hybridization, that is that only the decidua made detectable prolactin, while it was undetectable in the culture medium from trophoblast tissue, irrespective of the stage of pregnancy. This study, for the first time, establishes that only decidualized cells are involved in biosynthesis of prolactin; other prolactin-containing cells in the amnion and trophoblast appear to sequester prolactin, possibly via receptors, suggesting that prolactin may play an important paracrine role within the amnion and syncitio- and cytotrophoblast of the utero-placental unit.