Folliculogenesis: Physiology and pathophysiology

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1 DIMITRIOS LOUTRADIS Professor of Obstetrics and Gynaecology Head of 1st Department of Obstetrics and Gynaecology University of Athens Medical School Alexandra Hospital Folliculogenesis: Physiology and pathophysiology Disclosure of Interest: Nothing to Disclose

2 The earliest descriptions of reproduction 5 th Century B.C. - Hipocrates Generation occurred through joining of the male ejaculate and female menstrual blood 15 th Century A.D. William Harvey Ex ovo omnia: All things come from the egg Short R, 1972 Cobb M, th Century A.D. Regnier de Graaf Ovary : the producer of eggs Cobb M, A.D. Karl Ernst von Baer Egg The follicle itself is not the egg. Oocyte is enclosed within the ovarian follicle Cobb M, 2006

3 Means for studying antral folliculogenesis Histologic Endocrinologic Ultrasonographic techniques In vitro growth and maturation of follicles

4 Folliculogenesis Folliculogenesis in human is much greater than 220 days or eight menstrual cycles McGee and Hsueh, Endocrin Rev, 2000

5 Duration of follicle recruitment McGee & Hsueh, Endocrine Reviews, 2000

6 Theories of antral follicle recruitment Antral follicles 2 5 mm develop continuously OR cohorts or waves of antral follicles develop in a cyclic manner during the menstrual cycle. However, there is inconsistency in the literature, regarding the number of recruitment episodes that occur during the human menstrual cycle

7 Theory I Continuous recruitment throughout the menstrual cycle independent of gonadotrophins The follicle destined to ovulate is said to be selected, by chance, from the continuous supply of antral follicles by virtue of being at the right stage of maturity to respond to the rise in FSH (and subsequently LH) that occurs following luteal regression McNatty, 1981 Westergaard et al., 1986 Baird, 1987

8 Theory II Recruitment of a cohort of antral follicles once in the lateluteal phase or early-follicular phase of each cycle

9 Theory III Recruitment of two or three cohorts or waves during each cycle

10 FSH threshold and window The selective rise in FSH levels that occurs during the luteal-follicular transition is a potent stimulus for follicle recruitment, and several early antral follicles begin to enlarge in this phase of the cycle

11 Duration of FSH rise The duration of the rise in FSH above a critical threshold determines the number of dominant follicles selected from the recruited cohort for preferential growth This concept has been termed the FSH Threshold/Window/Gate Concept The duration that FSH is above the threshold is short for the development of a single dominant follicle The future dominant follicle may contain more granulosa cells and FSH receptors, making it more sensitive to FSH, compared with the subordinate follicles

12 Estradiol By Day 5 8 of the menstrual cycle, aromatase activity begins in granulosa cells of follicles larger than 6 8 mm, with the dominant follicle producing more estradiol-17β than other follicles in the cohort Estradiol-17β produced by the dominant follicle provides negative feedback on pituitary FSH secretion, which contributes, in part, to the midfollicular phase decrease in circulating FSH and inhibition of subordinate follicle growth LH receptors form on granulosa cells of the dominant follicle following estradiol secretion. The dominant follicle therefore becomes less dependent on FSH and more responsive to LH during the selection process

13 Factors affecting folliculogenesis Cortvrindt and Smitz, Reprod Domest Anim, 2001

14 Key-regulator of folliculogenesis: The Oocyte (?) Oocyte-granulosa cell interaction The key-regulator of folliculogenesis is probably the oocyte, as it secretes soluble factors with paracrine properties, which regulate granulosa cell proliferation and differentiation. The latter in turn regulate oocyte growth and maturation

15 Kit-Ligand & c-kit (Stem cell factor or Steel factor) Kit-Ligand: Soluble factor derived from granulosa cells c-kit or Stem cell factor or Steel factor: Tyrosine kinase receptor of the platelet-derived growth factor receptor family located on oocytes and theca cells. Promotes oocyte and follicle development It has been suggested that the oocyte promotes the production of Kit-Ligand by the granulosa cells, which in turn binds oocyte c-kit promoting oocyte

16 TGF-β Superfamily Inhibin Activin Growth differentiation factors: GDF-9 Bone morphogenetic proteins: BMP-15 ή GDF-9B AMH

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18 Growth differentiation factor-9 (GDF-9) Homodimeric protein of the TGF-β/activin family that presumably signals via serine-threonine kinase receptors In follicles it is expressed exclusively in the oocyte throughout folliculogenesis McGrath et al, Mol Endocrinol, 1995 Participates via autocrine effects in the regulation of oocyte development and maturation and via paracrine effects in the regulation of granulosa cell proliferation and differentiation Dong et al, Nature, 1996 Elvin et al, Mol Endocrinol, 1999 Promotes follicle survival and development through the suppression of cell apoptosis and follicle atresia Orisaka et al, Mol Endocrinol, 2006

19 Bone morphogenetic protein BMP-15 (GDF-9B) It is expressed exclusively in the oocyte from primary to preovulatory stage Aaltonen et al, J Clin Endocrinol Metab, 1999 Galloway et al, Nat Genet, 2000 Stimulates granulosa cell proliferation mediated by FSH Hayashi et al, Endocrinology, 1999 Otsuka et al, J Biol Chem, 2000 Vitt et al, Biol Reprod, 2000 Inhibits luteinization Shimasaki et al, Endocr Rev, 2004 Inhibits progesterone production Otsuka et al, J Biol Chem, 2000

20 Inhibin/Activin

21 Inhibin/Activin Inhibin B Decrease the FSH that occurs prior to selection Inhibin A Increases LH-induced thecal androgen production, which was then thought to serve as the substrate for dominant follicle estradiol production Activin B Stimulates FSH production from the gonadotropes by autocrine/paracrine mechanisms Activin A Inhibitory effect on LH-induced production of progesterone production i.e. inhibits spontaneous luteinization in mature antral follicles attenuates LH-dependent androgen production

22 Anti-Mullerian Hormone (AMH) Intrafollicular AMH gradually decreases during antral follicular growth until 8 10 mm (i.e. approximate time of selection), when AMH profoundly decreases and remains low thereafter.the decrease in intrafollicular AMH during selection of the ovulatory follicle occurs in association with a rise in granulosa cell aromatase expression

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25 CRH (Corticotropin Releasing Hormone) 41-amino acid neuropeptide The gene of CRH was first isolated from the hypothalamus of the sheep and is located at chromosome 8, region 8q13.1

26 Receptors CRH-R1 & CRH-R2 The two distinct receptors mediate the biological effects of CRH CRH-R1 CRH-R2 Share at 70% identical sequences and have many different isoforms They interact with G-proteins and consist of 7 transmembrane regions of a-helix

27 Antagonist of the receptor CRH-R1: Antalarmin Non-peptide antagonist of CRH Shows high affinity to CRH-R1 Has been used in the study of the role of CRH in stress, in inflammation reactions and in reproduction Suppresses the pathways that lead to stress without suppressing the activity of the HPA axis which would cause metabolic problems and other side effects Chrousos et al., 1998 Makrigiannakis et al., 2001 Kalantaridou et al., 2004 Ζoumakis et al., 2006

28 2 η Day ημέρα 0 Day 2 Day 4 Day 6

29 Day 8 Day 10

30 After 10 days of culture, ovulation was triggered by injecting 1.5 IU/ml hcg and 5 ng/ml EGF in the medium Samples of 500 μl of spent medium were collected sequentially on culture days 3, 5, 7, 9 and 11. In addition, samples of culture medium were also collected on day 1 for baseline analysis Glycoprotein hormone β-hcg Steroid hormones Estradiol Progesterone The results of the hormones were analyzed by employing ANOVA followed by post-hoc tests (Dunnett s test for comparisons with a control Bonferroni test for multiple comparisons).

31 RNA extraction and cdna preparation Total RNA extracted from preantral follicles obtained sequentially on culture days 3, 5, 7, 9 and 11 as well as morula and blastocyst stage development Total RNA extracted was reverse transcripted and cdna was prepared

32 Experimental groups for evaluation of early embryo development (i) Control group in vitro culture in the basic culture medium (ii) CRH 10-7 mol/l group in vitro culture in the basic culture medium supplemented with CRH in a concentration of 10-7 mol/l (iii) CRH 10-7 /antalarmin 10-6 mol/l group in vitro culture in the basic culture medium supplemented with CRH in a concentration of 10-7 mol/l and antalarmin in a concentration of 10-6 mol/l

33 Evaluation of CRH and its inhibitor, antalarmin, on in vitro growth of preantral follicles and early embryo development Preantral Maturation 2-cells p- 4-cells p- 8-cells p- M/B N N / % N / % value N / % value N / % value N / % p- value Control / / / / / 8.94 CRH , / / 9.94 < / 7.60 < / 3.80 < / CRH 10-7 / antalarmin , / / NS 59/ NS 44/ NS 34/ 9.82 NS CRH 10-7 vs CRH 10-7 / antalarmin 10-6 <0.001 <0.001 <0.001 <0.001

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37 Expression of CRH-R1 & CRH-R2 According to the quantitative analysis, we found that mouse preantral and antral follicles express both CRH-R1 and CRH-R2, although CRH-R2 is expressed to a lesser extent. We also found that mouse preimplantation embryos of the morula/blastocyst stage express CRH-R1 but not CRH-R2 mrna.

38 In conclusion Our data suggest a new role for CRH in follicular development, which is reversed by its inhibitor antalarmin. This role is characterized by a reduction in steroidogenesis, resulting from a direct and or indirect, through IL-1 or/and IGF-1 pathway, effect of CRH on the granulosa-lutein cells. Our findings demonstrate a new mechanism involved in oocyte maturation during in vitro follicle growth (IVG) conditions driven by CRH, which interferes with nuclear maturation, as previously shown (CRH inhibits in vitro oocyte maturation in mice. Fertil Steril 2011,95: ), but also seems to affect cytoplasmic maturation. In particular, the specific antisteroid (antiestrogen) action of CRH alters oocyte s microenvironment with regard to the inductive signals needed to complete cytoplasmic maturation, a mechanism that is reversible by antalarmin. This novel evidence of action in cytoplasmic maturation could offer the basis for further research regarding the physiology of folliculogenesis, especially in IVG cultures.

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41 Corticotropin-releasing hormone exerts direct effects on neuronal progenitor cells: implications for neuroprotection Y Koutmani, PK Politis, M Elkouris, G Agrogiannis, M Kemerli, E Patsouris, E Remboutsika and KP Karalis Molecular Psychiatry (2013) 18, & 2013 In this study they found that CRH could reverse the damaging effects of glucocorticoid on neural stem/progenitor cells (NS/PCs), while its genetic deficiency results in compromised proliferation and enhanced apoptosis during neurogenesis. Analyses in fetal and adult mouse brain revealed significant expression of CRH receptors in proliferating neuronal progenitors. On the other hand our study could serve the basis for further research to enrich our knowledge regarding the physiology of folliculogenesis process enhance fertilization, cleavage rates of the developing embryos and as well as to use as a tool for the development neural stem cells.

42 Growth Hormone (GH), PRL, & Insulin-like Growth Factor-I (IGF-I)

43 Local regulation of ovarian function by steroid and non-steroid agents has recently been described: Growth Hormone (GH) Insulin-like Growth Factors (IGFs) Epidermal Growth Factor (EGF) Neuropeptides, such as Prolactin (PRL) Many of these agents are produced by the follicle itself and have either an autocrine or paracrine mechanism of action Endocrine Paracrine Autocrine Intracrine For this purpose we have demonstrated in our laboratory the beneficial effects of GH, IGF-1 and PRL on in vitro maturation of mouse DOs

44 Effects of GH, IGF-I and PRL on in vitro maturation of mouse oocytes Substance studied GH (Lilly) Human PRL (Sigma Chemical Co.) Human IGF-1 (R & D Systems) Concentration 0.1, 0.2, 0.4, 0.6 μg/ml 100, 200, 400, 500, 600 ng/ml 10, 50, 100 and 200 ng/ml Collected oocytes were rinsed twice in a DPBS-BSA mixture solution (9:1) and cultured in Ham s F-10 medium without hypoxanthine (GIBCO BRL), supplemented with BSA (9:1) Approximately, 1,600 mouse DOs were obtained. Oocytes were isolated at the late stages of development being devoid of layers of cumulus cells and having germinal vesicle clearly visible

45 Figure 1: Oocytes at the late stages of development, devoid of layers of cumulus cells and having germinal vesicle clearly vidsible. The oocytes were collected from the oviducts of 2- to 8-week old female mice, without hormonal ovarian stimulation. The germinal vesicles of the oocytes are visible (black arrow) (magnification, x30) Figure 2: Effects of GH on in vitro maturation of mouse DOs. The percentage of cultured oocytes extruding first polar body after 72 hours (maturation rate), in the presence or absence (control) of GH (μg/ml) is presented. (a significantly different from the control; p<0.01) a a

46 a a Figure 3: Effects of IGF-1 on in vitro maturation of mouse DOs. The percentage of cultured oocytes developing a polar body after 72 hours (maturation rate), in the presence or absence (control) of IGF-1 (ng/ml) is presented. (a significantly different from the control; p<0.01) Figure 4: Effects of PRL on in vitro maturation of mouse DOs. The percentage of cultured oocytes developing a polar body after 72 hours (maturation rate), in the a b b b presence or absence (control) of PRL (ng/ml) is presented. (a, b significantly different from the control; p<0.05 and p<0.01 respectively)

47 a a a a a Figure 5: Effects of different combinations of GH (μg/ml), IGF-1 (ng/ml) and PRL (ng/ml) on in vitro maturation of mouse DOs. The percentage of cultured oocytes extruding first polar body after 72 hours (maturation rate), in the presence or absence (control) of the growth factors is presented. (a significantly different from the control; p<0.01)

48 Conclusions The observations recorded that the combined utilization of PRL and either IGF-1 or GH in the in vitro maturation of mouse DOs, suggest the existence of a common or related mechanism of action between PRL and either IGF-1 or GH In addition, certain relations between their mechanisms of action have been recognized. In fact, PRL appears to hold an important role in the IVM process, by enhancing the effects of GH and IGF-1 on the IVM of DOs, at least in certain concentrations

49 Prolactin receptor mrna expression in oocytes and preimplantation mouse embryos RBM Online Vol 10. No Reproductive BioMedicine Online; on web 24 January 2005

50 Aim To elucidate the role of Prolactin in IVM and in the process of reproduction and early embryo development in mice, we examined the expression of the four transcript variants of Prolactin Receptor PRL-RL PRL-RS1 PRL-RS2 PRL-RS3 in o Cumulus cells o Oocytes o Zygotes o 2-cell embryos o 4-cell embryos o Morula o Blastocyst

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53 PRL receptor mrna expression mrna Oocytes Cumulus cells Zygotes 2-cell embryos 4-cell embryos morulae Blasto cyst PRL-RL PRL-RS PRL-RS PRL-RS

54 Effect of PRL on In Vitro Follicle Growth, In Vitro Oocyte Maturation, Fertilization and Early Embryonic Development in Mice Cloning ans Stem Cells Volume 11, Number 2, pages , 2009

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56 Aim To elucidate the role of Prolactin in IVM and in the process of reproduction in mice, we examined the effect of this hormone in in vitro follicle growth and in the expression of the four transcript variants of prolactin receptor PRL-RL PRL-RS1 PRL-RS2 PRL-RS3 in opreantral follicles ocumulus-oocytes complexes (COCs) ocumulus separated oocytes (GV)

57 Materials & Methods Early preantral follicles 100μm-130μm were obtained from ovaries of 14-day old female mice, which were sacrificed by cervical dislocation In vitro follicle culture was performed for 12 days in α-mem in the presence of recombinant human FSH, insulin, transferrin, and selenium, supplemented with 5% fetal calf serum Substance studied Prolactin Concentration 20 ng/ml 100 ng/ml 200 ng/ml 300 ng/ml On day 12 of in vitro culture, recombinant hcg plus human EGF were added to the culture medium

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61 PRL receptor mrna expression mrna Preantral COCs (GV) PRL-RL PRL-RS PRL-RS PRL-RS

62 Discussion Female knock-out mice for PRL-LR, which overexpress a transgenic construct of PRL-S1 isoform, present follicular impairment, follicular degeneration, and follicular cell death. This impairment is due to the short PRL isoform activation, as wild-type mice, or females heterozygous for the PRL-S1 short isoform gene, exhibit normal follicular development. It seems that the long PRL isoform expression reverses the negative effect of the short isoform on follicles. According to our results, the PRL-RS1 is not expressed in any of the evaluated stages. Halperin et al., 2008 Sharov et al., 2003

63 Discussion PRL-R is located on chromosome 15, among the genes predominantly expressed in mouse stem cells and early embryos, such as LIF-R, EGF-R, GDF-9, and kit ligand. Matoba et al., 2006 PRL-R expression is one of the primary targets of Oct3/4, a transcription factor that plays an important role in maintaining pluripotency in both mouse and human embryonic stem cells. Sharov et al., 2003

64 Conclusions This study demonstrates that different isforms of PRL-R are present in early preantral follicles, COCs, and GV-stage oocytes. It seems that prolactin acts on its receptor in early preantral follicles, in COCs, and in GV-stage oocytes, resulting in increased nuclear maturation rates. The cleavage and early embryonic development rates are augmented when preantral follicles are cultured in the presence of PRL. This observation can be helpful in better understanding mechanisms that regulate follicle development from preantral follicles, and emphasizes the need to include PRL in experimental protocols designed to develop oocytes from cultured embryonic stem cells.

65 Take home messages Folliculogenesis is a quite complex process Several factors are implicated in this process In vitro follicle growth (IVG) and in vitro maturation (IVM) studies may serve in the identification of novel factors affecting folliculogenesis as well as the elucidation of the exact role of already known factors which are involved

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