Selçuk ÖZDEMİR, Selim ÇOMAKLI, Harun ARSLAN

Holştayn Sığırlarında Foliküler ve Luteal Fazdaki Ovaryum Dokularında İntraovarian Genlerin Ekspresyon Profili

Bu çalışmanın amacı, Holştayn sığırlarına ait preovülatör folikül ve korpus luteum dokularında BMP15, TGFB1, TGFB2 ve GDF9 genlerinin ekspresyon seviyelerini karşılaştırılmalı olarak belirlemektir. Bu amaç için, öncelikle dokular immunohistokimyasal boyama ile incelendi. Daha sonra foliküler sıvılar ELISA testiyle incelenerek östradiol ve progesteron seviyeleri belirlendi. Son olarak qRT-PCR ile gruplar arasında ilgili genlere ait ekspresyon seviyeleri tespit edildi. İmmunohistokimyasal boyama sonucunda preovülatör foliküllerde yoğun miktarda östrojen reseptör alfa ve progesteron reseptör immunpozitifliklerine, korpus luteum da çok hafif düzeyde östrogen alfa reseptör immunpozitifliğine, progesteron reseptörlerinin ise preovülatör foliküllerdeki pozitiflik düzeyine yakın olduğu belirlendi. Östradiol seviyesi, preovulatör foliküllerde yüksek, progesteron seviyesi ise korpus luteumda yüksek olarak bulundu. Preovülatör foliküllerdeki TGFB1 ve TGFB2 genlerine ait mRNA transkript seviyesi korpus luteuma göre istatistiksel olarak daha yüksek bulundu (p<0.01, p<0.05, sırasıyla), ancak gruplar arasında BMP15 ve GDF9 genlerine ait mRNA transkript seviyesinde istatistiksel olarak bir fark gözlenmedi (p>0.05). Sonuç olarak intraovarian genlerin farklı ekspresyonunun, ovaryum dokusunu oluşturan hücre popülasyonu içinde folikül dinamikleri ve gen ekspresyon seviyelerinin farklılıklarıyla ilişkili olabileceğini ve bu durumun da foliküler ve luteal dönemin sonucu olarak ortaya çıkabileceği düşünülmektedir.

Anahtar Kelimeler:

Korpus luteum, holştayn, preovulatör folikül, , intraovarian genler, qRT-PCR

Expression Profile of Intraovarian Genes in Ovary Tissues at Follicular and Luteal Phases in Holstein Cattle

The aim of the present study is to determine comparatively expression levels of the BMP15, TGFB1, TGFB2 and GDF9 genes in the preovulatory follicle and corpus luteum tissue of Holstein cattle. For this purpose, primarily the tissues were examined by immunohistochemical staining. Later on, follicular fluid was analyzed by ELISA test and estradiol and progesterone levels were determined. Finally, expression levels of the related genes were determined between the groups by qRT-PCR. Immunohistochemical staining revealed that estrogen receptor alpha and progesterone receptor immunoreactivities were intensely present in preovulatory follicles, estrogen alpha receptor immunoreactivity was very slight and progesterone receptors were similar to positivity in preovulatory follicles in corpus luteum. Furthermore, estradiol level was high in preovulatory follicles and progesterone level was high in corpus luteum. The levels of mRNA transcripts of the TGFB1 and TGFB2 genes in the preovulatory follicles were statistically higher than the corpus luteum (p<0.01, p<0.05, respectively), but there was no statistically significant difference between the groups in the mRNA transcript levels of the BMP15 and GDF9 genes (p>0.05). As a result, it is thought that differential expression of intraovarian genes may be associated with differences in follicular dynamics and gene expression levels within the cell population of ovarian tissue, so this situation may result in follicular and luteal phase.

Keywords:

Corpus luteum, holstein, intraovarian genes, preovulatory follicle, qRT-PCR,

PDF

___

Ball PJH, Peters AR. Reproduction in Cattle. Third Edition, 242 p, Blackwell Publishing, Ltd, 2004; 9600 Garsington Road, Oxford OX4 2DQ, UK.
Barros CM, Satrapa RA, Castilho AC, Fontes PK, Razza EM, Ereno RL. Effect of superstimulatory treatments on the expression of genes related to ovulatory capacity, oocyte competence and embryo development in cattle. Reprod Fertil Dev. 2012; 25: 17–25.
Berisha B, Pfaffl MW, Schams D. Expression of Estrogen and Progesterone Receptors in the Bovine Ovary During Estrous Cycle and Pregnancy. Endocrine. 2002; 17(3): 207–214.
Bliss SP, Navratil AM, Xie J, Roberson MS. GnRH signaling, the gonadotrope and endocrine control of fertility. Front Neuroendocrinol. 2010; 31(3): 322–340.
Burrow HM. Importance of adaptation and genotype x environment interactions in tropical beef breeding systems. Animal. 2012; 6(5): 729–740.
Corduk N, Abban G, Yildirim B, Sarioglu-Buke A. The effect of vitamin D on expression of TGF beta1 in ovary. Exp Clin Endocrinol Diabetes. 2012; 120(8): 490–503.
Delman HD, Eurell JA. Textbook of Veterinary Histology. 5th ed., 252- 325, 1988; Williams-Wilkins, London.
Donadeu FX, Pedersen HG. Follicle development in mares. Reproduction in Domestic Animals. 2008; 43: 224–231.
Drummond AE, Findlay JK. The role of estrogen in folliculogenesis. Mol Cell Endocrinol. 1999; 151: 57-64.
Farberov S, Meidan R. Thrombospondin-1 Affects Bovine Luteal Function via Transforming Growth Factor-Beta1-Dependent and Independent Actions. Biol Reprod. 2016; 94(1): 25.
Gilchrist RB, Morrissey MP, Ritter LJ, Armstrong DT. Comparison of oocyte factors and transforming growth factor-b in the regulation of DNA synthesis in bovine granulosa cells. Mol Cell Endocrinol. 2003; 201(1–2): 87–95.
Graham JD, Clarke CL. Physiological action of progesterone in target tissues. Endocr Rev. 1997; 18: 502-519.
Hanukoglu I. Steroidogenic enzymes: structure, function, and role in regulation of steroid hormone biosynthesis. J Steroid Biochem. 1992; 43(8): 779–804. Huias-Stasiak M, Gawron A. Immunohistochemical localization of estrogen receptors ER alpha and ER beta in the spiny mouse (Acomyscahirinus) ovary during postnatal development. J Mol Hist. 2007; 38: 25-32.
Juengel JL, Bibby AH, Reader KL, Lun S, Quirke LD, Haydon LJ. The role of transforming growth factor-b (TGF-b) during ovarian follicular development in sheep. Reprod Biol Endocrinol. 2004; 2: 78–88.
Juengel JL, McNatty KP. The role of proteins of the transforming growth factor-beta superfamily in the intraovarian regulation of follicular development. Hum Reprod Update. 2005; 11(2): 143–60.
Knight PG, Glister C. TGF-beta superfamily members and ovarian follicle development. Reproduction. 2006; 132(2): 191–206.
Lee WS, Otsuka F, Moore RK, Shimasaki S. Effect of bone morphogenetic protein-7 on folliculogenesis and ovulation in the rat. Biol Reprod. 2001; 65(4): 994–9.
Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and The 2 (-Delta Delta C(T)) Method. Methods. 2001; 25: 402–408.
Matiller V, Stangaferro ML, Díaz PU, Ortega HH, Rey F, Huber E, Salvetti NR. Altered expression of transforming growth factor-beta isoforms in bovine cystic ovarian disease. Reprod Domest Anim. 2014; 49(5): 813-23.
Merk FB, Botticelli CHR, Albright JT. An intercellular response to estrogen by granulosa cells in rat ovary: An electron microscope study. Endocrinology. 1972; 90: 992-1007.
Nagashima T, Kim J, Li Q, Lydon JP, DeMayo FJ, Lyons KM. Connective tissue growth factor is required for normal follicle development and ovulation. Mol Endocrinol.2011; 25(10): 740–59.
Otsuka F. Multiple endocrine regulation by bone morphogenetic protein system. Endocr J. 2010; 57(1): 3–14.
Otsuka F, Moore RK, Shimasaki S. Biological function and cellular mechanism of bone morphogenetic protein-6 in the ovary. J Biol Chem. 2001; 276(35) :32889–95.
Oullette Y, Price CA, Carrière PD. Follicular fluid concentration of transforming growth factor-beta1 is negatively correlated with estradiol and follicle size at the early stage of development of the first-wave cohort of bovine ovarian follicles. Domest Anim Endocrinol. 2005: 29(4); 623–33.
Panoulis K, Christantoni E, Pliatsika P, Anagnostis P, Goulis DG, Kondi-Pafiti A, Armeni E, Augoulea A, Triantafyllou N, Creatsa M, Lambrinoudaki I. Expression of gonadal steroid receptors in the ovaries of post-menopausal women with malignant or benign endometrial pathology: A pilot study. Gynecol Endocrinol. 2015; 31: 613-617.
Paradis F, Novak S, Murdoch GK, Dyck MK, Dixon WT, Foxcroft GR. Temporal regulation of BMP2, BMP6, BMP15, GDF9, BMPR1A, BMPR1B, BMPR2 and TGFBR1 mRNA expression in the oocyte, granulosa and theca cells of developing preovulatory follicles in the pig. Reproduction. 2009; 138(1): 115–29.
Rao JU, Shah KB, Puttaiah J, Rudraiah M. Gene expression profiling of preovulatory follicle in the buffalo cow: effects of increased IGF-I concentration on periovulatory events. PLoS ONE. 2011; 6 e20754.
Saragueta PE, Lanuza GM, Baranao JL. Autocrine role of transforming growth factor b1 on rat granulosa cell proliferation. Biol Reprod. 2002; 66(6): 1862–8.
Shimasaki S, Moore RK, Otsuka F, Erickson GF. The bone morphogenetic protein system in mammalian reproduction. Endocr Rev. 2004; 25(1): 72–101.
Van den Hurk R, Zhao J. Formation of ovarian follicles and their growth, differentiation and maturation within ovarian follicles. Theriogenology. 2005; 63(6): 1717–1751.
Weller MM, Fortes MR, Porto-Neto LR, Kelly M, Venus B, Kidd L, do Rego JP, Edwards S, Boe-Hansen GB, Piper E, Lehnert SA, Guimarães SE, Moore SS. Candidate Gene Expression in Bos indicus Ovarian Tissues: Prepubertal and Postpubertal Heifers in Diestrus. Front Vet Sci. 2016; 18: 3-94.
Wolfler MM, Kiippers M, Rath W, Buck VU, Meinhold-Heerlein I, Classen-Linke I. 2016. Altered expression of progesterone receptor isoforms A and B in human eutopic endometrium in endometriosis patients. Ann Anat. 2016; 206: 1-6.