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The reproduction and development is a complex and prolonged process. In mammals it involves oogenesis and spermatogenesis followed by fertilisation, implantation and development. Prior to the above mentioned steps in females during birth, all the oocytes are arrested in the early stage of meiosis I in the prophase. Shortly before the maturation process meiosis resumes and germinal vesicle breakdown occurs. The oocyte during ovulation would found to be arrested in the metaphase of the meiosis II. In humans even after fertilisation and implantation the embryos are arrested i.e., there is no gene transcription and thus no protein production until the 6 - cell stage (Zhang, Carrillo & Darling 1997).
It is proposed that there are a number of autocrine, paracrine and endocrine factors that help regulate the process of follicular development and ovulation. Many gonadotrophins and signalling pathways are also involved in the switching of genes where and when necessary. To mention a few are steroids, growth factors and prostaglandins.
Thyroid hormones have critical effects on the development process. It has been thought that Tri-iodothyronine (T3) might have important role in the development of ovarian follicle and also in the maturation of the Cumuls-oocyte complex (COC). Early in 1992, it was proved that administration of T3 enhanced the effectiveness of clomiphine which induced ovulation in amenorrhoea condition. Hypothyroidism leads to disruption of normal ovarian function, and could lead to polycystic ovarian syndrome in either adults or in young girls. Moreover, T3 has also shown to stimulate human granulosa cell proliferation and to synergize human chorionic gonadotrophin in in vitro conditions.
In mammals, the expression of T3 is regulated by the thyroid hormone receptor. The three isoforms of thyroid hormone receptor TRÎ±-1, TR Î²-1 and TR Î²-2 regulate the transcription of the target genes thus helps mediate T3 responses. In addition to the isoforms there is c-erbAÎ±-2 which is a spliced product of TR Î± gene that acts as a ligand-independent inhibitor for thyroid hormone in case of over-expression. The regulation could be explained using the response of T3 in the brain as an example. In developing brain the T3 response is high which is related to the increased expression of TR Î², whereas the absence of T3 response in an adult brain is due to the increased expression of the inhibitor c- erbAÎ±-2.
Scientists as early as 1993 have detected the TR isoforms TRÎ±-1 and TR Î²-1 in human mural granulosa cells. It was also found later that the oocytes and cumulus cells express TR mRNA and that the follicular fluid has high amount of T3 which has significant effects on the oocyte and cumulus cells. In case of Porcine follicles thyroid hormone in the form of T4 and T3 when supplemented with the ooctyes alone did not show significant influence but when used with combination with FSH in order to have direct stimulatory effects on not only the enhancing the granulosa cell function but also morphological differentiation, LH/human CG receptor formation and steroidogenic enzyme (3Î²-hydroxysteroid dehydrogenase and aromatase) induction (Maruo et al. 1987)
The effect of T3 on Mice oocytes were studied and it was found that there was no adequate influence on the maturation of the Cumulus oocyte complex (COC) and the meiotic resumption so as to facilitate the progress of oocytes beyond the metaphase II of meiosis. This was found to be similar for both mature and immature mice. However the T3 supplementation in vitro found to have negative effects on the Follicle stimulating hormone (FSH) aromatase activity, which lead researchers to believe that oocyte TR mRNAs might have been stored to help in the early oogenesis process as formerly seen in amphibian oocytes, eg. Xenopus laevis. The decrease in the FSH-induced aromatase activity in mature mice was induced sooner i.e., within 2 days of incubation, whereas in immature mice oocytes it took 6 days (Cecconi et al. 1999).
In addition to Thyroid Hormone and TR, Thyroid stimulating hormone is also found to have significant role in the reproduction and development process. Thyroid Stimulating hormone Receptor (TSHR) which belongs to the subfamily of G-protein coupled receptors similar to that of Follicle Stimulating Hormone Receptor and Luteinizing Hormone Receptor (LHR). But unlike the later FSHR and LHR which are expressed in gonads, TSHR is expressed mainly in the thyroid follicular cells, where upon TSH binding thyroid hormone is expressed through a series of 3'-5'-cyclic adenosine monophosphate (cAMP) cascade. Though the use of TSHR is known in thyroid follicular cells, TSHR is also found in other mammalian issues like lymphocytes, brown adipose tissues, erythrocytes, follicular-stellate cells, rectoocular fibroblasts, osteocytes, hepatocytes, neuronal cells and astrocytes, in most of which potential role have yet to be investigated (Sun et al. 2010).
The study of the influence of TSHR in mouse oocytes in MEM media with and without gonadotrophins showed that the combination of TSH and FSH induced expansion of cumulus cells (P<0.05), whereas LH failed to induce cumulus expansion (P>0.05) (De Silva, Pearl & Butler 1994). Similar research has also been carried out in bovine not only in the maturation stage but also in embryos until morula / blastocyst stage. It was found that oocytes Cumulus expansion (CE) was higher in the oocytes with hormones than the control that did not have any hormones. Despite the results of CE all the oocytes were then inseminated with heparin - capitated serum and the results were favourable for TSH and LH induced oocytes rather than those of the control and those subjected to Prolactin (PRL), thus proving the positive effect of TSH and LH on bovine oocytes (Younis, Brackett 1992).
Thyrostimulin a potent glycoprotein hormone is a found to be 30 fold more potent ligand for TSHR than TSH in studies made in rats. It is also proposed that there might be a paracrine action between thyrostimulin and TSHR in the ovarian compartments rather than the relationship between TSHR and TSH in itself. The fact regarding the paracrine and/or autocrine activity of thyrostimulin is yet to be proved through further experiments in the various compartments of the ovary. In addition to its significant presence in the female reproductive organ thyrostimulin is also found along with TSHR in testis of vertebrates as well (Sun et al. 2010).
In this study sheep is to be considered the existence of TR, TSHR, thyrostimulin and TH are to be analysed first in the whole ovaries obtained at different stages of the estrous cycle. The total RNA is to be isolated using the QIAGEN RNA preparation kit. The Total RNA is then to be subjected to reverse transcription with reverse transcriptase enzyme, oligo dT primers and dNTPs. The cDNA thus produced is subjected to PCR reaction with specific primers to detect the existence of TR, TSHR, TH and thyrostimulin. The PCR product was the run on agarose gel with controls to check the existence of mRNA and if positive further compartmentalisation of the ovary or immunohistochemical analysis to confirm further.
Cecconi, S., Rucci, N., Scaldaferri, M.L., Masciulli, M.P., Rossi, G., Moretti, C., D'Armiento, M. & Ulisse, S. 1999, "Thyroid hormone effects on mouse oocyte maturation and granulosa cell aromatase activity", Endocrinology, vol. 140, no. 4, pp. 1783-1788.
De Silva, M., Pearl, A.W. & Butler, W.J. 1994, "Thyroid stimulating hormone causes cumulus expansion in mouse oocytes", Theriogenology, vol. 41, no. 4, pp. 899-905.
Maruo, T., Hayashi, M., Matsuo, H., Yamamoto, T., Okada, H. & Mochizuki, M. 1987, "The role of thyroid hormone as a biological amplifier of the actions of follicle-stimulating hormone in the functional differentiation of cultured porcine granulosa cells", Endocrinology, vol. 121, no. 4, pp. 1233-1241.
Sun, S.C., Hsu, P.J., Wu, F.J., Li, S.H., Lu, C.H. & Luo, C.W. 2010, "Thyrostimulin, but not thyroid-stimulating hormone (TSH), acts as a paracrine regulator to activate the TSH receptor in mammalian ovary", The Journal of biological chemistry, vol. 285, no. 6, pp. 3758-3765.
Younis, A.I. & Brackett, B.G. 1992, "Thyroid stimulating hormone enhancement of bovine oocyte maturation in vitro", Molecular reproduction and development, vol. 31, no. 2, pp. 144-151.
Zhang, S.S., Carrillo, A.J. & Darling, D.S. 1997, "Expression of multiple thyroid hormone receptor mRNAs in human oocytes, cumulus cells, and granulosa cells", Molecular human reproduction, vol. 3, no. 7, pp. 555-562.