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" Characterization and regulation of the rat ovarian LHbeta mRNA "
Naomi Litichever
Y. G. Koch, Irith
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Latin Dissertation
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English
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| Record Number
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52425
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TL22379
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3461769
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| Main Entry
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Naomi Litichever
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Characterization and regulation of the rat ovarian LHbeta mRNA\ Naomi Litichever
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| College
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The Weizmann Institute of Science (Israel)
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| Date
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2008
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Ph.D.
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2008
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84
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| Abstract
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The hypothalamic gonadotropin releasing hormone (GnRH) is the main regulator of reproduction. However, the whole spectrum of hormones that are involved in the regulation of reproduction are present in all the levels of the reproductive axis: the hypothalamus, pituitary and ovary. The ovary produces a wide range of hormones that are currently known to regulate reproduction and their receptors too, including: GnRH and its receptor, estrogen and progesterone and their receptors, as well as activin, inhibin, follistatin, prolactin and their receptors. In addition, both gonadotropic hormones, LH and FSH were found to be produced also in the human, rat and mouse testes, and in the gilthead seabream oocytes. Therefore we decided to find out whether the rat ovary expresses LH and FSH, which together with the existence of GnRH might constitute a local hormonal circuit within the ovary. In this study we demonstrate that indeed, the rat ovary expresses LHβ, FSHβ and the common α-subunit mRNAs. In addition we quantified by radioimmunoassay the amount of LH in the ovary and demonstrated that the pituitary is not the source of ovarian LH. We hypothesized that ovarian LH might play a role in the rat pregnancy, but we could not find any indication that the ovarian LHβ mRNA or the ovarian LH content are increased during pregnancy. However, we found that the pituitary LHβ mRNA decreased during the second half of the pregnancy, while the pituitary LH content increased, suggesting that pituitary LH release decreases during this time. Indeed, serum LH concentrations decline towards the end of pregnancy. In addition we found that while ovarian and pituitary GnRH mRNA expression decrease during pregnancy, hypothalamic GnRH mRNA expression increased at the beginning of pregnancy, just before the time when LH is critical. This suggests that also during this critical period of pregnancy when LH is essential, pituitary LH release is regulated by hypothalamic GnRH. In parallel, we examined the regulation of LHβ and of GnRH mRNA in-vitro, in granulosa cell culture, and found that granulosa cells isolated from PMSG primed rats expressed lower LHβ and GnRH mRNA concentrations as compared to cells purified from estrogen primed rats. We speculated that the effect of PMSG is through the FSH receptor, and indeed, FSH reduced the expression of LHβ mRNA in granulosa cells from estrogen primed rats. However, FSH had no effect on the expression of GnRH mRNA. These results are in accord with our in-vivo findings that ovarian LHβ mRNA expression is reduced after PMSG treatment and at estrous morning after the surge of both LH and FSH. LH treatment vastly increased the expression of GnRH mRNA of granulosa cells from PMSG primed rats, whereas the expression of LHβ had not changed. In contrast to the effect of GnRH on the expression of LHβ mRNA in the pituitary, GnRH agonist treatment resulted in a decrease in LHβ expression in granulosa cells from either estrogen or PMSG primed immature rats. The GnRH receptor of granulosa cells did not desensitize after GnRH agonist treatment, as antagonist treatment, in contrast to agonist treatment, did not affect the LHβ expression. The GnRH mRNA expression, however, was increased after GnRH agonist treatment only in mature granulosa cells derived from PMSG treated rats. However, other hormonal treatments, such as incubation of the granulosa cells with prolactin, progesterone or estrogen did not result in any change in the expression of LHβ or GnRH mRNAs. These results indicate that the expression of LH in the ovary is specific and dependent on the maturation state of the follicle and is regulated by locally produced GnRH and by pituitary/ovarian LH and FSH. Thus, the hormonal regulation of gene expression in granulosa cell cultures depends both on the expression of the relevant receptor and on the maturational state of the cells. One can not conclude from one model of granulosa cells (i.e. from estrogen primed rats) what changes would occur in the other model (granulosa cells derived from MSG primed rats). Ovarian LHβ mRNA, similar to testicular LH mRNA, includes a 1.9kb 5' UTR. In this UTR there is a translation start site that is functional when the sequence is transfected into 293T cells, as found by using a GFP fusion protein. The 36kD protein predicted to be encoded in the 5'UTR is highly conserved between the rat, mouse and the human genome, and is always located upstream of the LHβ gene. However this family of proteins do not resemble any other known protein, nor do they contain a known motif or block. On the contrary, they are predicted to be natively unfolded (unstructured), and are probably involved in translation or regulation of translation. Although the novel protein coded on the ovarian LHβ mRNA can be detected by an antiserum directed against a representative peptide derived from the sequence when this mRNA is expressed in 293T cells, we could not detect a protein of the same size in the ovary/testis. In a radioimmunoassay system this antiserum identified a relevant protein in the testis of adult rats. The detection of the ORF protein by this antiserum was slightly influenced by phosphatase treatment of lysates from 293T cells expressing the protein. However phosphatase treatment of ovarian lysates did not expose any new relevant band. In addition, we could not detect the expected 36kD protein in cultures of rat immortalized granulosa and theca cells, even though the cells express the long LH mRNA transcript. A protein of about 49kD was specifically recognized by our antiserum, but no peptide corresponding to the 36kDa protein was found on that gel when analyzed by mass-spectrophotometry. We have also shown that rat immortalized theca cells, in contrast to 293T cells, do not express the expected 36kD protein when transfected with the rat LH mRNA. However the EGFP-fusion protein can be expressed in these cells. This suggests that endogenously the ovarian LHβ mRNA area coding the predicted ORF is not detected by our antibody because the protein is either translated and post-transcriptionaly modified/degraded or the protein is not translated at all due to a suppressing mechanism, i.e. micro-RNA. To summarize, we showed that the rat ovary contains a hormonal circuit that includes both the gonadotropic hormones and GnRH. There is a feedback system within the ovary between these hormones, as one hormone influences the expression of the other. Although the amount of these hormones in the ovary is very small, this may be sufficient for the autocrine/paracrine activity within the follicle itself. Because FSH treatment reduced the expression of LHβ mRNA, we can assume that the main site of action of LH will be in the primordial or primary follicles.
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Biological sciences; Gonadotropic hormones; Gonadotropin releasing hormone; Granulosa cells; Molecular biology; Neurosciences; 0307:Molecular biology; 0317:Neurosciences
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Y. G. Koch, Irith
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The Weizmann Institute of Science (Israel)
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