Improvement of the developmental capacity of oocytes from prepubertal cattle by intraovarian IGF-I application

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Aus dem Institut für Tierzucht (Mariensee)

der Bundesforschungsanstalt für Landwirtschaft (FAL) Braunschweig

Improvement of the developmental capacity of oocytes from prepubertal cattle by intraovarian IGF-I application

I N A U G U R A L-D I S S E R T A T I O N zur Erlangung des Grades eines

D O K T O R S D E R V E T E R I N Ä R M E D I Z I N (Dr. med. vet)

durch die Tierärztliche Hochschule Hannover

Vorgelegt von

Armando José Oropeza Delgado aus Boraure/Venezuela

Hannover 2004

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Wissenschaftliche Betreuung: Apl.-Prof. Dr. H. Niemann

1. Gutachter: Apl.-Prof. Dr. H. Niemann

2. Gutachter: Prof. Dr. B. Meinecke

Tag der mündlichen Prüfung: 24.05.2004

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"La educación e instrucción pública son el principio más seguro de la felicidad general y la más sólida base de la libertad de los pueblos."

Simón Bolívar (Libertador de Venezuela)

17 de Septiembre 1819

To my family

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Contents

1 INTRODUCTION ... 1

2 REVIEW OF LITERATURE ... 4

2.1 Oogenesis... 4

2.1.1 Maturation of oocyte... 6

2.1.1.1 Nucleus and cytoplasmic maturation... 7

2.1.1.2 The nucleolus... 9

2.1.2 Developmental differences of oocytes derived from prepubertal calves and adult cows... 12

2.2 Folliculogenesis... 18

2.2.1 Morphology of follicular development... 18

2.2.2 Hormonal regulation of follicular development... 21

2.2.2.1 Role of growth hormone in oocyte and follicle development... 23

2.2.2.2 Role of the insulin-like growth factor-I in oocyte and follicular development... 25

2.2.2.3 Role of insulin-like growth factor binding proteins (IGFBPs)... 30

2.2.3 Follicular dynamics and endocrine changes in prepubertal calves... 31

2.2.4 Follicular dynamics in adult cows... 34

2.3 In vivo recovery of bovine cumulus oocyte complexes ... 38

2.3.1 Laparoscopic and laparotomic oocyte recovery... 38

2.3.2 Transvaginal oocyte recovery... 39

2.4 Gene expression ... 42

2.4.1 Gene expression in the eukaryotic cells... 42

2.4.2 Protein synthesis... 48

2.4.3 Gene expression in preimplantation embryos... 53

2.4.3.1 Activation of the embryonic genome... 53

2.4.3.2 Expression of developmentally important genes in preimplantation development... 55

2.4.3.2.1 Glucose transporters (Gluts), Glucose transporter -1 (Glut-1)... 55

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Contents

2.4.3.2.2 Eukaryotic translation initiation factor (eIFs), eukaryotic initiation

factor 1A (eIF1A)... 61

2.4.3.2.3 Transcription factors (TIFs), Uptream Binding Factor (UBF)... 62

2.4.4 Methodology for detection of mRNA gene expression... 64

2.4.4.1 Reverse Transcription-Polymerase Chain Reaction (RT-PCR)... 65

2.4.4.1.1 Reverse Transcription... 65

2.4.4.1.2 Polymerase Chain Reaction (PCR)... 66

2.4.4.2 Quantification of mRNA expression... 69

2.4.4.2.1 Semi-quantitative RT-PCR... 69

2.4.4.2.2 Real-time RT-PCR... 70

3 MATERIALS AND METHODS ... 74

3.1 Oocyte donors... 74

3.2 Treatments... 74

3.2.1 Controls (acetic acid 10 mM)... 74

3.2.2 Recombinant bovine somatotropin (rbST)... 74

3.2.3 Insulin-like growth factor I (IGF-I)... 74

3.2.3.1 Dilution of IGF-I and intraovarian injection... 75

3.3 Transvaginal-guided ovum pick up (OPU)... 78

3.3.1 Classification of cumulus-oocyte complexes... 79

3.4 In vitro embryo production ... 81

3.4.1 Preparation of the media... 81

3.4.1.1 PBS medium (Flushing medium)... 81

3.4.1.2 TCM air medium... 81

3.4.1.3 Maturation medium (TCM + BSA)... 81

3.4.1.3.1 Hormones eCG and hCG... 82

3.4.1.4 Fertilization media... 82

3.4.1.4.1 Sperm-TALP medium... 82

3.4.1.4.2 Fert-TALP medium... 82

3.4.1.5 Capacitation agents... 83

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Contents

3.4.2 In vitro maturation... 84

3.4.3 In vitro fertilization... 84

3.4.3.1 Preparation of semen... 84

3.4.4 In vitro culture... 85

3.4.5 Evaluation and freezing of embryos for RT-PCR analysis... 86

3.5 Determination of the relative abundance of Glut-1, eIF1A and UBF... 88

3.5.1 Isolation of mRNA for semi-quantitative RT-PCR... 88

3.5.1.1 Solutions used to isolate mRNA from embryos... 88

3.5.1.2 Isolation of mRNA from embryos... 89

3.5.1.3 Reverse transcription (RT)... 89

3.5.1.3.1 Solutions and reagents... 89

3.5.1.3.2 Preparation of reaction mixture and reverse transcription... 90

3.5.1.4 Polymerase Chain Reaction (PCR)... 91

3.5.1.4.2 Primers used for PCR... 92

3.5.1.4.3 Preparation of the reaction mixture for PCR... 92

3.5.1.4.4 Optimization of the PCR parameters for each gene... 94

3.5.1.4.5 Determination the linear range of amplification for each gene... 94

3.5.1.5 Analysis of the RT-PCR products by agarose gel electrophoresis... 95

3.5.1.5.2 Gel electrophoresis... 95

3.6 Experimental design ... 96

3.7 Statistical analysis ...100

4 RESULTS ...101

4.1 Optimization of the PCR parameters...101

4.2 Optimization of the semi-quantitative RT-PCR assay...101

4.3 Calves 6-7 months of age...104

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Contents

4.7 General aspects of oocyte development in calves and cows ...111

5 DISCUSSION...123

6 CONCLUSIONS ...133

7 SUMMARY ...134

8 ZUSAMMENFASSUNG...138

9 REFERENCES ...143

10 APPENDIX...211

10.1 Composition of fertilization media ...211

10.2 Composition of SOF medium...211

11 LIST OF TABLES...214

12 LIST OF FIGURES ...215

13 LIST OF ABBREVIATIONS...218

14 ACKNOWLEDGEMENTS...221

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Introduction

1 Introduction

The onset of puberty is characterized by changes in plasma concentration of growth hormone (GH) and changes in plasma and follicular fluid concentration of insulin-like growth factor I [IGF-I] (SIMPSON et al. 1991; ARMSTRONG et al. 1992b). Both molecules play an important role during follicular and embryonic development (HERRLER et al. 1994; GONG 2002). Recently, it has been demonstrated that increased follicular IGF-I levels via intrafollicular rh-IGF-I injection are associated with follicle selection in heifers (GINTHER et al. 2003c).

The use of oocytes from calves in in vitro embryo production programs bears considerable potential for an accelerated genetic gain in domestic livestock production through a reduced generation interval (LOHUIS 1995; ARMSTRONG et al. 1997). Ultrasound-guided ovum pick-up (OPU) via transvaginal follicular aspiration is a reliable technique for harvesting immature bovine oocytes repeatedly from live donors (PIETERSE et al. 1988; BUNGARTZ et al. 1995). Previously, OPU was adapted for the repeated non-invasive collection of oocytes from calves and heifers (RICK et al. 1996; KUWER et al. 1999). However, despite progress in the past few years, the rate of viable blastocysts derived from prepubertal donors is low in comparison with their adult counterparts (PRESICCE et al. 1997; KUWER et al.

1999; STEEVES et al. 1999). Differences between oocytes from calves and cows have been found with regard to size, ultrastructure, metabolism and cytoplasmic maturation (DE PAZ et al. 2001; DUBY et al. 1996; STEEVES and GARDNER 1999;

SALAMONE et al. 2001). With respect to metabolism, oocytes and embryos from calves show a delayed uptake of glucose and pyruvate and a reduced protein synthesis (STEEVES and GARDNER 1999; SALAMONE et al. 2001; GANDOLFI et al. 1998). In early bovine embryos, glucose uptake increases markedly between the 8 and 16-cell stage, coinciding with the activation of the embryonic genome, compaction and blastulation (RIEGER et al. 1992a; RIEGER et al. 1992b; KHURANA and NIEMANN 2000; TELFORD et al. 1990). Glucose is taken up into the cell by an energy dependent mechanism (Sodium-Glucose co-transporter SGLT) and with the aid of facilitative glucose transporters (Gluts 1-12) (PANTALEON and KAYE 1998;

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Introduction

AUGUSTIN et al. 2001). IGF-I and GH affect Glut-1 mRNA expression and glucose uptake in mouse embryos (PANTALEON and KAYE 1998; ZHOU et al. 2000).

Messenger RNA for GH and IGF-I receptors have been identified in bovine cumulus cells, oocytes and embryos (IZADYAR et al. 1997a; YASEEN et al. 2001).

Supplementation of embryo culture media with GH or IGF-I improved oocyte maturation and blastocyst yields indicating that these molecules play an important role in bovine embryonic development (HERRLER et al. 1992; IZADYAR et al. 2000;

SIRISATHIEN et al. 2003). However, the effects of an IGF-I application in vivo on bovine oocyte and embryo developmental competence have not yet been studied.

The gene for Glut-1 is expressed in bovine oocytes from adult donors and throughout preimplantation development. Expression is significantly affected by culture conditions (WRENZYCKI et al. 1999). It is higher in the trophectoderm (TE) than in the inner cell mass [ICM] (WRENZYCKI et al. 2003). Expression of Glut-1 in embryos derived from prepubertal calves and its relationship with the developmental competence of oocytes have not been studied.

RNA and protein synthesis as well as reversible changes in the phosphorylation of specific proteins are required for germinal vesicle breakdown (GVBD) during bovine meiotic maturation (HUNTER and MOOR 1987; SIRARD et al. 1989; KASTROP et al. 1990; CHIAN et al. 2003). It has been shown that protein synthesis is reduced in oocytes and cumulus cells from calves (GANDOLFI et al. 1998), whereas the nuclear maturation rates of oocytes from calves are similar to those of cows after 24 h of in vitro maturation (STEEVES and GARDNER 1999). Low levels of transcriptional and translational activity have been detected already in zygotes and 2-4-cell stage embryos (VIUFF et al. 1996; MEMILI and FIRST 1999). However, the major activation of the bovine embryonic genome occurs at the 8-16-cell stage (TELFORD et al. 1990) which is correlated with an increased protein synthesis and changes in chromatin structure due to acetylation of core histones (FREI et al. 1989; KASTROP et al. 1990; MEMILI and FIRST 1999). In mouse embryos, insulin and IGF-I stimulate protein synthesis (HARVEY and KAYE 1988; HARVEY and KAYE 1991). Eukaryotic translation initiation factor 1A [(eIF1A), formerly called eIF-4C], is a transiently expressed endogenous marker of genome activation found in mouse and bovine

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Introduction

embryos (DAVIS, JR. et al. 1996; DE SOUSA et al. 1998b). Amongst others, eIF1A is involved in the initiation of translation in eukaryotic cells (CHAUDHURI et al. 1997) mainly by catalyzing the transfer of Met-tRNAfeIF2-GTP complex to the 40S ribosomal subunits to form a stable 40S preinitiation complex thereby accomplishing 60S subunits during translation (CHAUDHURI et al. 1999). Upstream binding factor (UBF) is involved in recognizing the rRNA gene promoter and activates RNA polymerase I mediated transcription (SCHNAPP et al. 1991). UBF plays a critical role in synthesis and assembly of the 60S and 40S eukaryotic ribosome subunits required for protein synthesis (LEARY and HUANG 2001). Expression of the mRNAs for eIF1A and UBF has not been studied in embryos derived from prepubertal calves.

The objective of the present study was to determine whether a systemic GH treatment and a local intraovarian IGF-I application can affect the developmental competence of oocytes from prepubertal calves. The expression pattern of three putative marker genes i.e. Glut-1, eIF1A and UBF was assessed in in vitro-produced embryos prior to and after major genomic activation. To determine putative changes in oocyte collection efficiency and oocyte developmental competence around the onset of puberty, oocytes were retrieved over a period of 7 – 8 months from the same animals starting at 6-7 months of age. Oocytes from adult cows were included in this study to compare treatment effects.

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Review of Literature

2 Review of Literature

2.1 Oogenesis

Oogenesis, the process of gamete formation in the female mammal, involves the proliferation, growth and maturation of oocytes, the formation and rupture of the follicle and the release of a mature ovum, i.e. ovulation (HAFEZ 1962). Oogenesis begins already during early fetal development with the formation of primordial germ cells (10-20 µm in diameter), which populate the primitive fetal ovary via independent amoeboid-like movement and in response to different substances such as transforming factor β1, and then divide to form ovarian stem cells [oogonia] (PICTON and GOSDEN 1998). In humans and other large mammalian species, the number of oogonia (12.5-25 µm in diameter) increases through proliferation and many rounds of mitotic divisions over a period of several months until approximately day 170 of gestation in cattle (ERICKSON 1966a; PICTON and GOSDEN 1998). No new oogonia are formed after birth (SOLOMON et al. 1996a). After several mitotic divisions, the oogonia increase in size and become primary oocytes. The oocytes progress to the first meiotic prophase, which is initiated between days 75 and 80 post conception in cattle (ERICKSON 1966a; LAVOIR et al. 1994). During prophase I genetic material is exchanged between homologous chromatids by crossing over, a process in which enzymes break the chromatids, exchange parts, and then rejoin the chromatids to produce new combinations of genes (SOLOMON et al. 1996a). The meiotic prophase is composed of several transitory stages: preleptotene, leptotene, zygotene, pachytene and the diplotene stage in which the first meiotic division in mammalian oocytes, including cattle oocytes is arrested. In this stage the chromosomes are uncondensed forming a vesicular nucleus, the so-called germinal vesicle (GV) stage, with abundant cytoplasmic organelles (BAKER and FRANCHI 1967; DOWNS 1993b).

The arrested primary oocytes constitute the terminally differentiated germ cells, which are surrounded by a layer of flattened granulosa cells to form the primordial follicle, that constitute the stock of non-growing follicles in the ovary (ERICKSON 1966a;

BRAW-TAL and YOSSEFI 1997; YANG et al. 1998). At birth calves have

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approximately 133,000 oocytes (ERICKSON 1966b), which remain arrested at the dictyate stage of meiosis.

The maintenance of meiotic arrest of mammalian oocytes is due to inhibitory effects exerted by components of the follicular fluid. Already PINCUS and ENZMANN (1935) showed that when germinal vesicle stage oocytes were removed from the environment of the antral follicle, they undergo spontaneous meiotic maturation in vitro. Two specific types of inhibitory molecules have been shown to be important in controlling meiotic maturation, cyclic nucleotides and purines. It has been demonstrated in rodents (SCHULTZ et al. 1983) as well as in bovine (AKTAS et al.

1995a; AKTAS et al. 1995b) that cyclic adenosine 3’,5’-monophosphate (cAMP) can block spontaneous meiotic maturation of oocytes in culture. cAMP analogs or phosphodiesterase (PDE) inhibitors (isobutyl methyl xanthine, IBMX) that prevent degradation of intracellular cAMP, reversibly suppress germinal vesicle breakdown (GVBD) in vitro (SIRARD and FIRST 1988). Recent reports suggest that the maintenance of meiotic arrest is regulated by the interplay of cAMP, cumulus, granulosa cells and theca cells (RICHARD et al. 1997; RICHARD and SIRARD 1998;

AKTAS et al. 2003).

Other cyclic nucleotides may be involved in the control of meiosis. Cyclic guanosine 3’, 5’-monophosphate (cGMP) analogs and microinjection of cGMP have been shown to inhibit spontaneous oocytes maturation (TORNELL et al. 1990).

Purines such as hypoxanthine and adenosine have been found at millimolar concentrations in preparations of follicular fluid that maintain oocytes in meiotic arrest in vitro, likely through suppression of cAMP degradation (DOWNS et al. 1989;

DOWNS 1993a; DOWNS 1999). A limited effect of purines has been reported in bovine oocytes (SIRARD 1990).

Although the oocytes remain arrested in prophase of the first meiotic division, RNA transcription of heterogeneous nuclear RNA (hnRNA, the precursor of mRNA) and rRNA and protein synthetic activity continue until the oocytes become meiotically competent (FAIR et al. 1997a). In bovine, the RNA synthesis appears to decrease when oocytes reach a diameter of about 110 µm or the follicle reaches a diameter of 3 mm (FAIR et al. 1995; FAIR et al. 1996). After this stage only a low level hnRNA

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synthesis is preserved (FAIR et al. 1995), which can be detected up to the GV stage while at metaphase II (M-II) a sharp decrease is observed (MEMILI et al. 1998). To prevent degradation of mRNAs and proteins during storage in the oocyte, post- transcription and post-translation mechanisms, such as mRNA polyadenylation and deadenylation described for oocytes of lower organisms, are also essential in the cow (BREVINI-GANDOLFI et al. 1999; GANDOLFI and GANDOLFI 2001).

The growth phase of the oocyte is associated with the formation of cumulus cell processes in the oolema, forming the gap junctions between the cumulus cell layer and the oocyte, which facilitate the transfer of signal molecules as well as nutrients between the oocyte and the cumulus cells (EPPIG 1991; FAIR et al. 1997b). During the growth phase, there is considerable modification of cytoplasmic organelles including the endoplasmic reticulum (ER) and the mitochondria and there is a development of oocyte-specific structures such as the zona pellucida and the cortical granules. These structures play an important role during fertilization when they are responsible for blocking of polyspermy. The zona pellucida is synthesized in the secondary follicle stage and is composed of three cross-linked, sulfated glycoproteins which are referred to as ZP 1, ZP 2 and ZP 3 (FAIR et al. 1997b; SINOWATZ et al.

2001).

FAIR et al. (1995) determined that oocytes at a diameter of about 100 µm, acquire the full competence for resumption of meiosis, and at a diameter of about 110 µm, reach the full competence for completing meiotic maturation to metaphase II.

However, cleavage and blastocyst rate increase significantly when oocytes with diameter higher than 120 µm are fertilized (DE LOOS et al. 1989).

2.1.1 Maturation of oocyte

Oocyte maturation is traditionally defined as those events associated with the initiation of germinal vesicle breakdown (GVBD) and completion of the first meiotic division, referred to as nuclear maturation. More correctly, oocyte maturation is defined as those events that render the oocytes capable for fertilization and initiate the program that directs preimplantation embryonic development (LEIBFRIED-

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RUTLEDGE et al. 1987). This process constitutes maturation of the nucleus and the cytoplasm (NIEMANN and MEINECKE 1993).

2.1.1.1 Nucleus and cytoplasmic maturation

During the oocyte growth phase, the inner zona diameter of the gamete increases from less than 30 µm in the primordial follicle to more than 120 µm in the tertiary follicle (BRAW-TAL and YOSSEFI 1997; HYTTEL et al. 1997). Resumption of meiotic maturation in vivo occurs in Graafian follicles in response to the preovulatory gonadotropin surge when puberty is reached (SCHAMS et al. 1981; ARMSTRONG 2001).

During meiotic maturation, major morphological changes occur, including germinal vesicle breakdown (GVBD), chromosome condensation, rearrangements of microtubule network, development of the lipid store, probably as an energy source for the initial phase of embryonic development, reduction of the Golgi compartment, spatial rearrangement of mitochondria and alignment of the cortical granules along the oolema, all of which are necessary to reach the first meiotic metaphase [M-I] (DE LOOS et al. 1991; HYTTEL et al. 1986a; HYTTEL et al. 1987; HYTTEL et al. 1989;

HYTTEL et al. 1997; KRUIP et al. 1983; ROZINEK et al. 1995; SUSS et al. 1988).

Oocytes then proceed directly through anaphase I and telophase I to produce two haploid cells of different size (BAKER and HUNTER 1978). The smaller one, the first polar body, may later divide, forming two polar bodies, but these eventually disintegrate. The large cell, the secondary oocyte, proceeds to the second meiotic division but remains in metaphase (M-II) until it is fertilized or parthenogenetically activated (SOLOMON et al. 1996a; LIU et al. 1998b). The ability of the oocyte to proceed through different events related to the nuclear and cytoplasmic maturation is not acquired in a single step. Initially, the oocyte acquires the ability to fully condense its chromatin and to break the nuclear membrane (GVBD). This requires mainly two factors to be activated. The activation of the first factor termed Maturation Promoting Factor or M-phase Promoting Factor (MPF), composed of p34^cdc2 and cyclin B (NURSE 1990), starts about 7 h after initiation of in vitro culture and occurs at two levels (KUBELKA et al. 2000). The first is association of p34^cdc2with the regulatory

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subunit cyclin B1 (LEVESQUE and SIRARD 1996). The second level is the phosphorylation and dephosphorylation of different sites on p34^cdc2 (ALBERTS et al.

1994d; NURSE 1990). This factor is thought to be crucial in regulating G2/M transition, not only in meiotic cells (oocytes) but also in somatic cells. The second factor is referred to Mitogen-Activated Protein (MAPK) kinase, a serine/threonine kinase. The activation of this factor starts in bovine oocytes about 8 after in vitro culture (KUBELKA et al. 2000), and requires phosphorylation on both tyrosine and threonine residues by an upstream kinase identified as a dual specific MAP kinase kinase (MAPKK), also termed MEK (MATSUDA et al. 1992; MATSUDA et al. 1993;

MEINECKE and KRISCHEK 2003). The temporal pattern of activation during resumption of meiosis suggests that MAPK activation may be linked to p34^cdc2 kinase activation (KUBELKA et al. 2000; MEINECKE and KRISCHEK 2003). A temporal pattern of inactivation has been also observed in fertilized or parthenogenetically activated oocytes, since inactivation of MAPK is preceding MPF inactivation (LIU et al. 1998a; MOOS et al. 1995). In Xenopus oocytes an activator of MAPK is the product of the proto-oncogene c-mos, a 39 kDa serine/threonine kinase known as Mos (POSADA et al. 1993). In cattle, the activation of MAPK by Mos RNA injection into oocytes and the detection of Mos protein in oocytes suggest the presence of a Mos/MAPK signal transduction pathway (FISSORE et al. 1996; WU et al. 1997).

RNA transcription and protein synthesis as well as reversible changes in the phosphorylation of specific proteins are required for germinal vesicle breakdown (GVBD) and reaching metaphase II during bovine meiotic maturation. HUNTER and MOOR (1987) observed that bovine cumulus-enclosed oocytes exposed to the RNA inhibitor alpha-amanitin or the protein synthesis inhibitor cycloheximide did not undergo germinal vesicle breakdown after 28-h of culture. Similarly, KASTROP et al.

(1991b) showed that mRNA transcription and protein synthesis are necessary for phosphorylation of specific proteins and for GVBD. Furthermore, transcription during the first hour of culture is needed for synthesis of new proteins after GVBD. SIRARD et al. (1989) demonstrated that protein synthesis is needed at four different steps (GVBD, chromatin condensation, metaphase I and metaphase II) in bovine oocyte maturation. Changes in the pattern of protein synthesis and the phosphorylation of

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three phospho-proteins are an important feature of maturation. The first protein has a molecular weight which is in the range of 19 to 24 kDa. The other two phospho- proteins have molecular weights of 50 and 60 kDa and all three phospho-proteins are found in oocytes which have undergone GVBD (KASTROP et al. 1990; KASTROP et al. 1991a). In a recent report, it was shown that reversible phosphorylation of proteins with molecular weights of 40, 27, 23 and 18 kDa may be related to either cell cycle transition or pronucleus formation during the maturation and fertilization in bovine oocytes (CHIAN et al. 2003).

In conclusion, the final maturation of the oocyte occurs after the LH surge and is characterized by expansion of the cumulus oophorus, disruption of the gap junctions, increase of lipid contents, reduction in the Golgi compartment, alignment of the cortical granules just inside the oocyte membrane, expulsion of the first polar body and arrest at metaphase of the second meiotic division (HYTTEL et al. 1986b;

HYTTEL et al. 1989; HYTTEL et al. 1997).

2.1.1.2 The nucleolus

Many of the maternally derived products needed to prepare the biological machinery, such as re-assembly of the nucleolus, as factory for ribosomes necessary for protein synthesis are stored in the oocytes during its growth (DIELEMAN et al. 2002). The nucleolus contains large loops of DNA emanating from several chromosomes, each of which contain a cluster of rRNA genes, which are transcribed by RNA polymerase I [Pol I] (ALBERTS et al. 1994e). Each such gene cluster is known as nucleolar organizer region (NOR), where pre-ribosomal RNA is synthesized, processed and assembled with specific proteins into pre-ribosomal particles (KING et al. 1988). The transcription of these genes is considered a prerequisite for the formation of the nucleolus that develops around the NORs (HYTTEL et al. 2000b; HYTTEL et al.

2001). The number of NORs, like the chromosome number, is characteristic of the species. The diploid bovine genome comprises ten NORs localized to the telomere regions of chromosome 2, 3, 4, 11 and 29 (DI BERARDINO et al. 1979).

The nucleolus is the most prominent structure within the eukaryotic cell nucleus and is the site where ribosomal RNAs (5.8S, 18S, and 28S) are transcribed by specific

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nucleolar transcription factors and enzymes. These transcripts become associated with specific ribosomal proteins to form ribosomal subunits (SCHWARZACHER and WACHTLER 1993; ALBERTS et al. 1994e). Subsequently the subunits leave the nucleus through the pores of the nuclear envelope and associate to form ribosomes in conjunction with translation of mRNAs (HYTTEL et al. 2000b). Growing cells require continuous ribosome synthesis to ensure that subsequent generations contain the ribosomes necessary to support protein synthesis. The actively ribosome- synthesizing nucleolus consists of three main components. The fibrillar centres (FCs), the dense fibrillar component (DFC) and the granular component (GC). The FCs are frequently situated in the central region of the nucleolus. The DFC forms a network of strands surrounding the FCs, but may sometimes stretch out towards the periphery of the nucleolus. The GC is usually situated in the peripheral regions of the nucleolus (SCHWARZACHER and WACHTLER 1993). These components of the so- called fibrillo-granular nucleolus reflect the steps in the biosynthesis of ribosomes according to the following model: The fibrillar centres house the enzymatic apparatus for the transcriptional process, the dense fibrillar component carries the primary unprocessed transcripts, whereas the granular component represent the processed transcripts associated with proteins in the form of preribosomal-particles (FAIR et al.

1996; HOZAK et al. 1994; HYTTEL et al. 2000b). It is believed that rRNA transcription occurs at the interphase between the fibrillar centre and the dense fibrillar component, and that the latter is formed by the newly synthesized rRNA (HYTTEL et al. 1997). In cells with a low level of ribosomal biosynthesis the nucleoli are small, usually with a single FC and little surrounding DFC and GC ("ring-shaped nucleolus"). In active cells the DFC forms a large network enclosing several, sometimes up to hundreds of FCs, and the GC covers a large area in the periphery ("compact nucleoli"). In cells at the onset of a new stimulation, the DFC is prominent whereas FCs are few and small, and the GC is not very extensive ("reticulate nucleoli") (SCHWARZACHER and WACHTLER 1993). Studies on involving embryos produced in vitro (IVP) have revealed that transcription of ribosomal RNA (rRNA) genes during embryo development occurs at a species-specific stage of preimplantation embryonic development (THOMPSON 1996). Nucleolar function is

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gradually acquired at follicle activation and the acquisition of fibrillar centres in oocyte nucleoli is accompanied by the formation of the antral cavity in early tertiary follicles (FAIR et al. 1997a). When the oocyte reaches approximately 110 µm in diameter, corresponding to a follicle of about 3 mm in diameter, transcription decreases and the nucleolus is inactivated, forming a dense spherical remnant [Fig. 1] (FAIR et al.

1996). During the final phase of follicular dominance this remnant becomes vacuolated and, in conjunction with resumption of meiosis, disperses. There are no nucleoli during or after germinal vesicle breakdown (HYTTEL et al. 2001). The formation of a fibrillo-granular nucleolus in the bovine embryo is a sequential process, which does not reach complete functional activity until late in the fourth cell cycle when the nucleolar proteins necessary for rRNA transcription and processing are assembled (LAURINCIK et al. 2000; VIUFF et al. 1998). In the pig, fibrillo- granular nucleoli are observed at the 4-cell stage in in vivo developed embryos (HYTTEL et al. 2000a) while in in vitro produced embryos it appears for first time at the 16-cell stage, displaying a substantial delay in nucleolar activation (BJERREGAARD et al. 2003). Bovine embryos produced in vitro and embryos developed in vivo display allocation of nucleolar proteins to fibrillar and granular compartments of the developing nucleoli during the fourth cell cycle (8-cell stage).

However, among in vivo developed embryos, the formation of tentative fibrillar centres might be seen as early as during the 2^nd cell cycle showing some differences with respect to the chronology of the nucleolar development. On the other hand, parthenogenetically activated bovine embryos display a delayed nucleolar activation, which occurs during the fifth cell cycle (HAY-SCHMIDT et al. 2001; LAURINCIK et al.

2003).

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Oocyte and follicle growth

primordial, primary, secondary and tertiary follicle

Fibrillar centres

Oocyte capacitation and maturation Resumption of meiosis LH

surge

<100 100 -110 >110 µm Oocyte size

Nucleolus

Dense nucleolar remnant Oocyte and follicle growth

primordial, primary, secondary and tertiary follicle

Fibrillar centres

Oocyte capacitation and maturation Resumption of meiosis LH

surge

<100 100 -110 >110 µm Oocyte size

Nucleolus

Dense nucleolar remnant

Fig: 1. Nucleolus ultrastructure during growth, capacitation and maturation of the bovine oocyte.

Fibrillar centres invade the granular nucleoli in the secondary follicle and are marginalized towards the end of oocyte growth with the formation of the dense nucleolar remnant as the result. The remnant is vacuolized towards the end of oocyte capacitation and is dispersed in conjunction with resumption of meiosis. There are no signs of the nucleolus during or after germinal vesicle breakdown. Adapted from HYTTEL et al. 2001.

2.1.2 Developmental differences of oocytes derived from prepubertal calves and adult cows

As in most mammalian species, in cattle folliculogenesis occurs during fetal development. Antral follicles are observed in fetal ovaries during late gestation and as many as 50 antral follicles appear when a calf reaches 2 months of age

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programs bears considerable potential for an accelerated genetic gain in domestic livestock production through a reduced generation interval (LOHUIS 1995; DUBY et al. 1996; ARMSTRONG et al. 1997). However, despite progress in the past few years, the rate of viable blastocysts derived from prepubertal donors is low in comparison with their adult counterparts (PRESICCE et al. 1997; STEEVES et al.

1999).

Most of the studies have reported that oocytes from young calves are deficient in their ability to develop to blastocysts and to produce successful pregnancies after embryo transfer in comparison with embryos derived from adult animal oocytes (PALMA et al. 1993; LOONEY et al. 1995; RICK 1996; KHATIR et al. 1998b). The rate of production for blastocysts was of 9-11% in nonstimulated and stimulated calves compared with 20% in adult cows (REVEL et al. 1995). Similarly, DAMIANI et al. (1996) found a low (6%) development rate to the blastocyst stage in oocytes from prepubertal calves in comparison with 33% for oocytes from adult cows. PRESICCE et al. (1997) showed that the developmental competence of oocytes from prepubertal calves is acquired with age. Oocytes from stimulated calves at 5 and 7 months of age had a lower rate of cleavage (24 and 49%) and development to blastocysts (0 and 17% / cleaved oocytes) than oocytes from adult cows (62% and 27%) for cleavage and blastocyst rate, respectively. However, when at 9 and 11 months of age these same animals (now pubertal heifers) were subjected to oocyte retrieval by ultrasound-guided ovum pick up (OPU) the rate of cleavage was not different to that of adult cows. Similarly, LOONEY et al. (1995) obtained a lower cleavage rate (33.8%) and a lower blastocyst rate (0%) for oocytes derived from prepubertal calves than for oocytes derived from the same heifers after they had reached puberty (63.4% and 18.9%) and for those from adult cows (73.3% and 31.6% for cleavage and blastocyst rate, respectively). In addition, STEEVES et al. (1999), determined that development to blastocysts of oocytes from 5 to 7 months old calves, was lower (9.8%) than of oocytes from adult cows (33.7%). In a recent study, CHOHAN and HUNTER (2004) observed a lower maturation rate (80.1% vs. 92.0%), fertilization rate (69.3% vs. 79.9%) and cleavage rate (36.7% vs. 49.9%) for oocytes derived from 7.5 months to term bovine fetuses than for those from adult cows, respectively.

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These results were attributed to the higher proportion of fetal oocytes (12%) which remained at the GV and metaphase I (M-I) stages than was observed for oocytes from adult cows after 24 h of in vitro maturation (2.3%). These studies show that the age of oocyte donor is a significant factor affecting the developmental competence of the oocyte. Differences between oocytes from calves and cows have been found with regard to size, ultrastructure, metabolism and cytoplasmic maturation (DUBY et al.

1996; DE PAZ et al. 2001; STEEVES and GARDNER 1999; SALAMONE et al.

2001).

Oocytes from prepubertal cattle were significantly smaller (113.3-119.7 µm) than those obtained from adult cows (117-125 µm), representing a difference of approximately 5% in diameter and 10% volume (DUBY et al. 1996; GANDOLFI et al.

1998; STEEVES and GARDNER 1999). Despite this fact, maturation rates after 24 hr of culture of oocytes from 4 and 7 months old calves (75-76%) and cows (78-86%) were similar (DAMIANI et al. 1996; STEEVES and GARDNER 1999). However, it has been clearly demonstrated that oocytes with a diameter >120 µm have a high maturation rate and developmental competence to form blastocysts (LONERGAN et al. 1994; FAIR et al. 1995).

Studies employing transmission electron microscopy reveled that oocytes postmaturation from calves showed a delay in migration and redistribution of the organelles as mitochondria, lipid vesicle and cortical granules, which remain associated in large aggregates instead of dispersing evenly below the plasma membrane, suggesting a compromised cytoplasmic maturation (DAMIANI et al.

1996). A lower mitochondrial population in calf oocytes than those from cows, suggests a lower energy metabolism in calf oocytes (DE PAZ et al. 2001).

GANDOLFI et al. (1998) observed that oocytes from 10- to 14-weeks old calves metabolized pyruvate and glutamine during the first 3 hr of in vitro maturation (IVM) at lower rates than adult oocytes. Similarly, STEEVES and GARDNER (1999) and GANDOLFI et al. (1998) reported a lower oxidative metabolism of pyruvate and glutamine and a delayed uptake of glucose in oocytes from 3-4 months old calves. In another report, STEEVES et al. (1999) observed a lower glucose uptake (1.5 pmoles/embryo/hr) in 2- to 4-cell stage embryos from calves 5-7 months of age than

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from their adult counterparts (3.0 pmoles/embryo/hr), but it was equivalent at 8- to 16-cell stage and blastocysts.

Metabolic studies of ovine oocytes also have demonstrated differences between oocytes derived from 6- to -8-week-old lambs and those from adult ewes (O'BRIEN et al. 1996). The metabolism of radiolabelled glutamine by prepubertal lamb oocytes was significantly lower than that of oocytes from adult ewes. The difference was observed irrespective of whether the oocytes were matured in vitro or in vivo. No differences were observed in other metabolic parameters including oxidation of glucose or pyruvate.

Reprogramming of protein synthesis plays an essential role in control of meiotic division, and in the preparation of the mammalian oocyte for fertilization. Synthesis of proteins appears to be compromised in calf oocytes. GANDOLFI et al. (1998) observed a significant decrease in protein synthesis, measured by [³⁵S] methionine and [³⁵S] cysteine incorporation after 9 hr of IVM in calf oocytes. Differences were also found in protein patterns. Thus, proteins of 405, 146, 101 and 77 kDa were more abundant in cow oocytes after of the first 3 hr of IVM than in calf oocytes. In a similar study, KHATIR et al. (1998a) determined significant differences in protein profiles of constitutive and de novo synthesized proteins during the first 20 hr of IVM, suggesting that protein synthesis could be a reason of the lower developmental competence of calf oocytes. Furthermore, LEVESQUE and SIRARD (1994) observed similar patterns of constitutive proteins in “defective” oocytes (oocytes with dark or expanded cumulus or not fully surrounded by cumulus cells) derived from cows and in oocytes derived from less than 40 day old calves. In this study, the absence of several constitutive proteins was also observed in cumulus cells from calf follicles.

These proteins may be important for the initiation of the cascade of events necessary for calf oocytes to develop normally. The existence of an oocyte–granulosa cell regulatory loop essential for normal follicular differentiation resulting production of an oocyte competent for fertilization and embryogenesis, has been proposed (EPPIG 2001). On the other hand, it has been reported that immature calf oocytes have a nucleolus that contains two electron-dense ovoid structures encapsulated by less electron dense fibrils, whereas immature cow oocytes possess only one structure

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(DAMIANI et al. 1996). The latter is characteristic of oocytes that have completed their RNA synthesis (FAIR et al. 1996). The presence of two fibrillar structures in calf oocytes may indicate incomplete nucleolar maturation (DAMIANI et al. 1996).

Biochemical changes and physiological events have been used as parameters to study cytoplasmic maturation in calf oocytes. Some examples are the activity of MPF and MAPK (involved in the resumption of meiosis) and the relative amount of inositol 1, 4, 5-triphosphate receptor (IP3R) which is involved in the generation of calcium oscillations during fertilization. A low activity of MPF and MAPK which can be measured by the amount of phosphorylation of histone H-1 kinase was observed.

The relative amount of IP3R as determined by Western blotting was lower in oocytes derived from 6 months old calves than in oocytes derived from adult cows (SALAMONE et al. 2001).

The rate of cleavage and the rate of blastocyst formation have been used to evaluate cytoplasmic maturation after nuclear transfer, after parthenogenetic activation and after stimulation by culture in follicular fluid. A lower blastocyst rate was obtained when nuclei derived from 30-50 cell morula stage embryos produced in vitro from adult oocytes were transferred to recipient ooplasm from prepubertal calf than when nuclei from the same embryos were transferred to ooplasm of oocytes collected from adult cows (MERMILLOD et al. 1998). Similarly, embryos reconstructed by fusion of M-II chromosomes from the oocytes of adult cows into calf ooplasm cleaved at a lower rate and produced fewer blastocysts than did those reconstructed by the transfer of M-II chromosomes from immature calves into adult ooplasm (SALAMONE et al. 2001). Similar results have been obtained with oocytes from prepubertal gilts (IKEDA and TAKAHASHI 2003). Both, cleavage and blastocyst rates were significantly lower in parthenogenetically activated calf oocytes than in activated adult cow oocytes (SALAMONE et al. 2001). Calf oocytes produced fewer blastocysts than cow oocytes in culture supplemented with cow follicular fluid or fetal calf serum, indicating that prepubertal oocytes are unable to respond or do not possess the receptors for specific factors present in serum and follicular fluid that probably stimulate the developmental competence in adult oocytes (KHATIR et al. 1997).

These findings suggest that cytoplasmic maturation is compromised in calf oocytes

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affecting the synthesis and storage of upstream regulators or key regulatory components for embryonic development.

No differences between calf and cow blastocysts have been observed with respect to constitutive protein synthesis, de novo protein synthesis or total cell number (KHATIR et al. 1998b; STEEVES et al. 1999). However, differences were observed in the viability of these embryos after transfer. Pregnancy rates of 0 to 22% have been reported after the transfer of blastocysts derived from calf oocytes as compared to 39% pregnancy after the transfer of blastocysts produced from adult cow oocytes (KHATIR et al. 1998b; RICK 1996). On the other hand, ARMSTRONG et al. (1997) reported an ultrasound confirmed 45-day pregnancy rate of 43% in 23 recipients of fresh IVF embryos from calf oocytes. These pregnancies eventually resulted in the birth of seven calves (33% of embryos transferred).

REVEL et al. (1995) reported that rates of initial pregnancies determined by progesterone level 21 days of the estrous cycle after transfer of IVF embryos produced from calf oocytes were similar to the rates obtained with adult cow oocytes, (66% and 65% respectively). However, only one of the nine recipients (11%) of embryos from the FSH treated calves maintained pregnancy and delivered a full-term calf indicating high embryo losses for blastocysts derived from prepubertal calves.

The low uptake of energy substrates, low activity of MPF and MAPK as well as low rate of protein synthesis indicate that cytoplasmic maturation with its essential biochemical changes is compromised in calf oocytes. This does not prevent completion of meiosis but does diminish the ability of these oocytes to produce blastocysts at rates observed for adult cow oocytes.

Age also affects the composition of follicular fluid. DRIANCOURT et al. (2001) determined a reduced level of estradiol, low aromatase activity and differences in protein profiles in follicular fluid of healthy follicles from 3 months old calves in comparison to those from cows. However, increased blastocysts yield of cow oocytes were found in cultures supplemented with calf follicular fluid (KHATIR et al. 1997).

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2.2 Folliculogenesis

2.2.1 Morphology of follicular development

Folliculogenesis begins with the formation of primordial follicles in the fetal ovary. The primary oocytes, which are arrested in the meiotic prophase, become surrounded by a squamous layer of pre-granulosa cells around day 90 of gestation to form the primordial follicles of the ovary (RÜSSE 1983). The factors that regulate primordial follicle formation are poorly understood. It has been demonstrated in mice that the c- kit receptor and its ligand play a central role in directing migration and survival of germ cell during embryonic life (DRIANCOURT et al. 2000). Expression of Figlα mRNA (also known as Figα), a gene that encodes a helix–loop–helix transcription factor which co-ordinates expression of structural genes for components of the zona pellucida, has been identified as a important factor during the formation of the primordial follicles (EPPIG 2001).

Primordial follicles constitute a population of resting, non-growing follicles which are progressively depleted during the reproductive life span. The depletion of the primordial follicle pool occurs as the result of two processes: Atresia mainly through apoptosis or entry into the growth phase (KAIPIA and HSUEH 1997; VAN VOORHIS 1998). Primordial follicles contain 4-8 flattened pre-granulosa cells and have a diameter of ~35 µm with an oocyte of approximately 30 µm in diameter [Fig. 2]

(BRAW-TAL and YOSSEFI 1997; FAIR et al. 1997b). Initiation of follicular growth involves the transition of primordial follicles from the quiescent to the growth phase and is characterized by three main events: change in shape of the granulosa cells from squamous to cuboidal, proliferation of granulosa cells and enlargement of the oocytes (BRAW-TAL and YOSSEFI 1997). The mechanism by which the primordial follicles are activated to start growth is still unclear. However, factors such as bone morphogenetic protein-7 (BMP-7), IGF-I, EGF, anti-mullerian inhibiting hormone (AMH), oocyte specific growth and differentiation factor-9 (GDF-9), BMP-2 receptor, BMP-15 and kit ligand are involved in this process, (for review see FAIR 2003).

Entry of the primordial follicle into the growth phase is characterized by conversion of the flattened of pre-granulosa cells surrounding the oocyte into a single layer of cuboidal granulosa cells (FAIR et al. 1997b). This follicle is now termed a primary

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follicle (VAN VOORHIS 1998). Primary follicles contain 8-20 cuboidal granulosa cells forming a single layer around the oocytes. The diameter is increased to ~55 µm, while size of the oocyte is not changed [Fig. 2] (BRAW-TAL and YOSSEFI 1997). In cattle, the first growing follicles appear in the fetal ovary around day 180 of gestation consisting mainly of primary and secondary follicles, the first antral follicles are observed approximately at day 220 of gestation (WANDJI et al. 1992).

Secondary follicles are characterized by a partial or complete double layer of cuboidal granulosa cells. Formation of the zona pellucida is associated with the formation of granulosa cells processes into the oolemma, forming gap junctions between the granulosa cells and oocyte (FAIR et al. 1997b). Theca interna cells can be recognized occasionally as elongated cells attached to the basement membrane (BRAW-TAL and YOSSEFI 1997). In primordial follicles the communication between the oocyte and the granulosa cells is apparently mediated through an endocytotic pathway as indicated by abundant coated pits and vesicles within the oocyte. In secondary follicles, communication is via gap junctions that are formed between the oocyte and granulosa cells. Tertiary follicles are characterized by antrum formation, a multilayer of granulosa cells surrounding the oocyte, formation of the basal lamina and the cumulus cells (FAIR et al. 1997b). These follicles have a clearly defined theca interna and externa and the oocyte is surrounded by a thick zona pellucida (BRAW-TAL and YOSSEFI 1997; FAIR et al. 1997b). Antrum formation starts in follicles ranging 0.14-0.28 mm. Growth of antral follicles can be divided in two phases. Firstly, early growth of follicles can be attributed to an increase in the number of granulosa cells and therefore an increase in the surface of granulosa layer. Secondly, in follicles larger than 2.5 mm, follicular growth results from antrum development rather than an increase of the number of granulosa cells (LUSSIER et al. 1987). The granulosa cells intimately surrounding the oocyte, constitute the cumulus oophorus, whereas the other granulosa cells are referred to as either antral granulosa cells (cells close to the antral cavity) or mural granulosa cells [cells close to the basement membrane of the follicle] (VAN VOORHIS 1998). The oocyte reaches a diameter of 92 µm in small antral follicles (BRAW-TAL and YOSSEFI 1997). In cattle the preovulatory or Graafian follicle is characterized by a diameter of 15 to 20

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mm, an oocyte with a diameter >120 µm, and an expanded cumulus oophorus due to the secretion of hyaluronic acid by the cumulus cells (EPPIG 2001). The extensions of the corona radiata penetrating the zona and reaching the oocyte are retracted to a more superficial position and are ultimately lost (HYTTEL et al. 1997). In this phase meiosis is resumed and the oocyte progresses through the final stages of meiosis I to be arrested at metaphase of meiosis II [Fig. 2] (HYTTEL et al. 1986b; LUSSIER et al.

1987). During the final stage of follicular development, transcription of specific genes, which are expressed transiently prior to follicle rupture, and after biochemical events cause an inflammatory-like reaction resulting in an increase in vasodilatation and hyperemia. The preovulatory follicle becomes a highly vascularized structure with exudation, edema, collagenolysis, cell proliferation, and tissue remodeling before rupture of the so-called follicular stigma is induced (ESPEY 1994; VAN VOORHIS 1998; RICHARDS et al. 2002). Ovulation occurs 25-30 hr after the LH surge (KAIM et al. 2003).

Fig: 2. Relationship between follicle development and oocyte growth in cattle.

Growth of oocytes (surrounded by granulosa cells) is shown schematically in relation to follicle diameter, oocyte diameter and estimated duration of stage of folliculogenesis from the primordial to preovulatory stage. Reproduced from FAIR 2003. Based on data from LUSSIER

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2.2.2 Hormonal regulation of follicular development

Ovarian follicular growth and development is a complex process encompassing both systemic extraovarian signals, such as gonadotropins and metabolic hormones (i.e.

growth hormone) and local intraovarian factors. Follicular development has been classified into gonadotropin-independent and gonadotropin-dependent phases (WEBB et al. 1999a). In the latter, Follicle-Stimulating Hormone (FSH) provides the primary trigger for follicular recruitment and Luteinizing Hormone (LH) is required for continued development of follicles to the preovulatory stage and induction of ovulation. FSH and LH are synthesized by specialized cells (gonadotropes) in the anterior lobe of the pituitary and released upon stimulation of the gland by the hypothalamic decapeptide gonadotropin-releasing hormone (GnRH) (REICHTER 1998). Gonadotropins are glycoprotein hormones with two oligosaccharide chains, linked to a free amide group of amino acids. FSH has a molecular weight about 30,000-70,000 and LH about 26,000-30,000 depending on the species (REIMERS 2003). They are composed of two noncovalently bound subunits. One is structurally similar for both hormones and the other one, the β subunit which is hormone specific.

FSH acts on the ovary to promote the development of primary follicles into large fluid- filled vascular preovulatory follicles (YING 1988). LH is associated with the phase of follicular dominance (GINTHER et al. 2001b) and stimulates the final maturation of the oocyte and ovulation (DIELEMAN et al. 2002). In the follicle, LH stimulates synthesis of androstenedione in the theca cells and FSH stimulates the activity of cytochrome P450 aromatase enzyme in the granulosa cells to convert androgens into estrogen (GINTHER et al. 2001a). During folliculogenesis intraovarian factors exert very important functions. For example, inhibin produced in large amounts by granulosa cells of the dominant follicle exerts a stimulatory paracrine action on androgen production (WRATHALL and KNIGHT 1995).

Figure 3 shows a summary of functions of different intraovarian factors during follicular development.

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Fig: 3. Diagram summarizing experimental observations on follicle stage-dependent expression of TGF-β superfamily members in ruminants and their putative roles as intrafollicular autocrine/paracrine signaling molecules.

Superscripts (o, g, and t) refer to oocytes, granulosa cells and theca cells, respectively. GDF growth differentiation factor, BMP bone morphogenetic protein, TGF transforming growth

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2.2.2.1 Role of growth hormone in oocyte and follicle development

Pituitary LH and FSH are known to be the major regulators of ovarian functions including folliculogenesis, steroidogenesis, and oocyte maturation. In past years, however, the biological relevance of GH for female reproductive physiology and its involvement in physiology of the ovary, including follicular growth and development were elucidated (GONG et al. 1993a; WEBB et al. 1994). GH and its receptor (GHR) belong to the large family of cytokine peptides and their receptors (BAZAN 1989). GH is a single-chain, 191 amino acid protein, mainly secreted by somatotropic cells located in the anterior pituitary gland as principal source, but it is also produced in brain, lymphocytes, placenta, mammary tissue, and the pineal gland, suggesting that GH also has local paracrine/autocrine effects possibly different from its classic effects that are mediated by circulating IGF-I (BUTLER and LE ROITH 2001). Growth hormone-releasing hormone (GHRH) and the inhibitory hormone Somatostatin (SRIF) interact to regulate GH secretion from the pituitary [Fig. 4] (BRAZEAU et al.

1973; SPIESS et al. 1983). Studies using RT-PCR and immunoblotting in ovaries from fetuses, prepubertal calves and adult cows have demonstrated the presence of mRNA encoding GHR and GHR protein in oocytes and granulosa cells which indicates that GH is involved in folliculogenesis (IZADYAR et al. 1997b; KOLLE et al.

1998). Furthermore, messenger RNA encoding GH and GH protein have also been found in immature and mature oocytes as well as in granulosa, theca and cumulus cells from antral follicles larger than 2 mm suggesting a paracrine and /or autocrine action of GH in bovine follicular growth (IZADYAR et al. 1999).

Recombinant bovine somatotropin (rbST) has been used in the last years in combination with FSH or eCG in superovulation protocols to increase the follicular number and total embryo yields. A increased number of small follicles (<5 mm) was obtained after single application of 320 mg rbST combined with eCG or aspiration of the dominant follicle in heifers at 2-3 years of age (BURATINI, JR. et al. 2000; GONG et al. 1993a; GONG et al. 1993b). Holstein dairy cows treated with rbST and eCG showed an increase in the number of large follicle (>4 mm) and number of corpora lutea (HERRLER et al. 1994). When 2.5 year old heifers were injected with a daily dose of 12.5 and 25 mg rbST over a period of 7 days, the number of ovarian follicles

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<5 mm in diameter, increased concomitantly with serum concentrations of IGF-I and insulin (GONG et al. 1997). Similarly, when calves were treated with recombinant bovine somatotropin in combination with FSH or eCG, the number of small follicles (<5 mm) and aspirated follicles was 1.3-1.6 times higher than in calves treated with FSH or eCG alone (HWANG et al. 1997). GH may thus be particularly important in the recruitment of small follicles. However, rbST does not affect the timing of the pattern of the follicular wave during the oestrus cycle, nor the inhibitory action of the dominant follicle on its subordinate follicles (GONG et al. 1993a).

YANG et al. (1998) injected six 9-9.5 month old heifers, which had a palpable corpus luteum (CL), with 500 mg rbST every two weeks for up to 8 times. Oocytes collected from these heifers by OPU had a higher developmental rate to the morula and blastocyst stage after in vitro fertilization than oocytes collected from age matched non-treated heifers. In contrast, BOLS et al. (1998) did not observe differences in the number of COCs recovered by OPU and the blastocyst rate in heifers treated weekly for 10 weeks with 640 mg of rbST.

The presence of mRNA encoding GHR and GHR protein in preimplantation bovine embryos has been determined (IZADYAR et al. 2000). Moreover, GH mRNA and pituitary-specific transcription factor-1 (Pit-1), that binds to promoters of the GH encoding gene, have been identified in 2-4-cell and 8-16- cell stage bovine embryos as well as in morulae and blastocysts (JOUDREY et al. 2003). The transcript for GHR has been identified in all stages of preimplantation mouse embryos and the transcript for GH in morulae and blastocysts (PANTALEON et al. 1997b). These observations suggest the possibility of a paracrine / autocrine GH loop regulating bovine embryonic development similarly as it has been proposed in mice.

It has been observed that addition of bovine growth hormone (bGH) to the culture medium accelerates in vitro maturation of cumulus-enclosed bovine oocytes, induces cumulus expansion, and promotes subsequent embryonic development in terms of increasing the number of blastocysts (IZADYAR et al. 1996; IZADYAR et al. 2000;

MOREIRA et al. 2002b).

Similar results have been obtained in vivo. Administration of 500 mg of rbST to superovulated Holstein cows in conjunction with a fixed artificial insemination

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decreased the number of unfertilized ova, increased the percentage of transferable embryos, and stimulated embryonic development to the blastocyst stage. Moreover, rbST increased the pregnancy rate following embryo transfer in recipients treated with rbST on day 1 after estrus (MOREIRA et al. 2002a). However, no differences in the pregnancy rate were observed when rbST was injected at the time of embryo transfer (HASLER et al. 2003). Obviously growth hormone can enhance embryo development by stimulation of proliferation and /or differentiation of embryonic cells as well as by modulating embryo metabolism. Growth hormone stimulates protein synthesis and glucose transport in mouse blastocysts (PANTALEON et al. 1997b).

This effect appears to be due to translocation of the glucose transporters from an intracellular position to the plasma membrane (TANNER et al. 1992).

2.2.2.2 Role of the insulin-like growth factor-I in oocyte and follicular development

The critical importance of IGF-I in follicular development is emphasized by the phenotype of Igf1 null mice whose follicles arrest at the late preantral/early antral stage and fail to respond to gonadotropins (ZHOU et al. 1997). The IGF family includes three known ligands (IGF-I, IGF-II, and insulin). Six characterized binding proteins (IGFBP-1 to -6) modulate their bioavailability and three cell surface receptors (IGF-I receptor, insulin receptor and the IGF-II mannose-6-phosphate receptor) mediate the action of the ligands (BUTLER and LE ROITH 2001). IGFBP-proteases are also included in the IGF family (RIVERA et al. 2001; GINTHER et al. 2003a).

Bovine insulin like growth factor I (IGF-I) is a 70 amino acid, single chain polypeptide with a molecular mass of 7649 daltons. The bovine cDNA is 93% identical to the human sequence and the amino acid sequence is 96% conserved (FOTSIS et al.

1990; SIMMEN 1991). IGF-I shares ~55% amino acid sequence homology with proinsulin and 62% with IGF-II (HAMMOND 1998; JONES and CLEMMONS 1995).

IGF-I is secreted mainly in the liver under the control of growth hormone [Fig. 4]

(HULL and HARVEY 2001). Thus, exogenous application of rbST increases the concentration of IGF-I in the peripheral blood and follicular fluid (GONG et al. 1991;

GONG et al. 1993b; HERRLER et al. 1994). IGF-I mRNA has been detected in the

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uterus, placenta, ovary, lung, kidney, heart, skeletal muscle, testes, brain and mammary gland of adult rats (MURPHY et al. 1987) and in the oviducts of buffalos and bovines (DALIRI et al. 1999; YASEEN et al. 2001). The messenger RNA for IGF- I has been detected in bovine immature oocyte and preimplantation embryos (WATSON et al. 1992; YOSHIDA et al. 1998) as well as at every developmental stage from oocyte to blastocyst in the mouse (DOHERTY et al. 1994). This suggests that oocyte derived IGF-I may have a function in follicular growth. In addition, messenger RNA for IGF-I receptor (IGF-IR) has been detected in immature and matured bovine oocytes (YASEEN et al. 2001), and in bovine granulosa cells, theca cells and oocytes from preantral and antral follicles (ARMSTRONG et al. 2000;

ARMSTRONG et al. 2002). Furthermore, bovine granulosa and theca cells isolated from antral follicles have been shown to synthesize IGF-I in vitro and IGF-I synthesis is affected by gonadotropins in the granulosa cells, but not in the theca cells (SPICER et al. 1993; SPICER and CHAMBERLAIN 2000). Porcine granulosa cells have been shown to synthesize IGF-I under the control of FSH and GH control (SAMARAS et al. 1996). Results of in vitro studies with bovine cells indicate that IGF- I increases the proliferation of follicle cells, enhances the sensitivity of granulosa cells to FSH, increases the secretion of estradiol, progesterone, inhibin-A, activin-A, follistatin and oxytocin from granulosa cells, and enhances LH-stimulated androstenedione and progesterone synthesis from theca cells (ARMSTRONG et al.

1996; SCHAMS et al. 1988; SPICER et al. 1993; SPICER and STEWART 1996;

WEBB et al. 1999b). In addition, in vitro culture of preantral bovine follicles has demonstrated a positive effect of IGF-I on follicle diameter and antrum formation as well on oocyte growth (GUTIERREZ et al. 2000; MCCAFFERY et al. 2001). An increased follicular IGF-I level after intrafollicular rh-IGF-I injection is associated with follicle selection in heifers (GINTHER et al. 2003c). Collectively, these findings demonstrate that IGF-I interacts synergistically with FSH and LH to regulate the proliferation and differentiation of granulosa and theca cells, and has a significant impact on follicular and oocyte development. The presence of mRNA for IGF-I receptors in the oocyte suggests that IGF-I could play an important role in the growth

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of the oocyte and accumulation of mRNAs necessary during early embryonic development. These actions may be both by endocrine and paracrine pathways.

In a recent study in mice, it was proposed that GH and IGF-I act synergistically but independently on cell proliferation and differentiation during embryo development.

GH mainly acts on the cells of the trophectoderm whereas IGF-I induces proliferation of the ICM through the IGF type 1 receptor (MARKHAM and KAYE 2003). The mRNA for the IGF-I receptor was detected in immature and mature oocytes and at all bovine embryonic stages (YASEEN et al. 2001). Culture of bovine COCs in the presence of IGF-I enhances the frequency of germinal vesicle activation and metaphase II indicating that this factor stimulates meiotic progression (LORENZO et al. 1995).

Furthermore, the findings from HERRLER et al. (1992) and PALMA et al. (1997) showed that culture medium supplemented with IGF-I combined with a monolayer of granulosa cells or estrous cow serum can improve the development of bovine embryos produced in vitro. Similarly, IGF-I in combination with epidermal growth factor (EGF) was shown to stimulate cumulus expansion, oxidative metabolism, nuclear maturation and cleavage after fertilization of bovine oocytes in vitro (RIEGER et al. 1998). Supplementation of the culture medium with IGF-I increased the proportion of human embryos developing to the blastocyst stage from 35% to 60%

(LIGHTEN et al. 1998). A concentration of 10 or 100 ng/ml of IGF-I in IVM medium stimulated meiotic maturation of both cumulus-enclosed and cumulus-free porcine oocytes (SIROTKIN et al. 1998). Supplementation of IVM medium with 100 ng/ml of IGF-I stimulated maturation of buffalo oocytes (PAWSHE et al. 1998). However, addition of IGF-I at a similar concentration did not improve nuclear or cytoplasmic maturation of sheep oocytes (GULER et al. 2000). IGF-I stimulates equine oocyte maturation in a dose-dependent manner with the highest nuclear maturation rate at a concentration of 200 ng/ml over a 36 hr incubation period, but no significant effect was observed after 48 hr (CARNEIRO et al. 2001). The addition of IGF-I to chemically defined in vitro culture medium (50 ng/ml) improved the proportion of COCs that reached blastocyst stage [38% vs. 28.5%] (SIRISATHIEN and BRACKETT 2003). Furthermore, supplementation of culture medium with IGF-I reduced the apoptotic index and elevated the total cell number in bovine blastocysts

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(BYRNE et al. 2002; MAKAREVICH and MARKKULA 2002). An increased number of inner cell mass (ICM) cells in bovine blastocysts was also observed at a concentration of 50 ng/ml of IGF-I in culture medium (SIRISATHIEN et al. 2003).

Effects of IGF-I on embryo development are mediated by stimulation of protein synthesis through the IGF-I receptor (HARVEY and KAYE 1991) and an increase of glucose uptake (PANTALEON and KAYE 1996) via expression and translocation of Glut-1 (PANTALEON and KAYE 1998; ZHOU et al. 2000). Insulin and IGF-I stimulate amino acid and glucose transport in blastocysts and placenta but IGF-I is 1000-fold more potent than insulin in the regulation of glucose transport in vivo (KNISS et al.

1994; PANTALEON and KAYE 1996).

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Hypothalamus

GHRH

⁺

SRIF

-

Liver

Pregnancy Uterus Ovary Anterior Pituitary

Hypothalamus

GHRH

⁺

SRIF

-

Liver

Pregnancy Uterus Ovary Anterior Pituitary

Fig: 4. Effects of GH in the female reproductive system.

Hypothalamic GHRH stimulates pituitary GH release and GH induces the production of hepatic IGF-I, and both pituitary GH and hepatic IGF-I act to stimulate mammary, ovarian, uterine and/or oviduct function. However, GH is also produced in the mammary gland, placenta, ovary (and possibly in the oviduct), and may act directly or via local IGF-I to affect reproductive function. Pituitary GH and hepatic IGF-I may be involved in normal regulation of ovarian function (italic text, solid lines), whereas ovarian GH may be involved in modulation of ovarian function at a critical point in follicular and oocyte development (normal text, dotted lines). Somatostatin (SRIF) inhibits GH release. Adapted from HULL and HARVEY 2001.

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2.2.2.3 Role of insulin-like growth factor binding proteins (IGFBPs)

IGF binding proteins (IGFBPs) regulate the bioavailability of IGFs during follicular development. IGFBP-1 to -6 mRNA has been determined in bovine theca cells and IGFBP-2 to -5 in bovine granulosa cells (SCHAMS et al. 1999). IGFBP-2 and 4 are produced by granulosa and theca cells in antral follicles (ARMSTRONG et al. 1998).

Furthermore, messenger RNA encoding for IGFBP-2 was detected in granulosa cells and oocytes of preantral follicles but IGFBP-3 mRNA was detected only in oocytes from preantral and antral follicles (ARMSTRONG et al. 2002). In cattle, IGFBP profiles are altered during follicular development, correlated with the follicular status.

IGFBP-3 concentrations remain unchanged in dominant follicles when compared with healthy subordinate follicles while IGFBP-2, -4 and -5 concentrations are significantly lower in dominant follicles than in subordinate follicles (AUSTIN et al. 2001;

FORTUNE et al. 2001; MIHM et al. 2000; SPICER et al. 2001). This is compatible with the observation that reduced intrafollicular concentrations of IGFBP-2 and -4 increase the availability of free IGF to enhance FSH responsiveness of the follicles, thus conferring a selective advantage on dominant follicle. These changes in the concentration of IGFBPs in follicular fluid occur through changes in gene expression (ARMSTRONG et al. 1998) and proteolysis (RIVERA et al. 2001). FSH causes inhibition of IGFBP mRNA expression in granulosa cells which results in decreased levels of IGFBPs in preovulatory follicular fluid (ARMSTRONG et al. 1998). In addition, it has been shown that injecting heifers with low doses of recombinant bovine FSH to induce co-dominant follicles triggers proteolysis of IGFBP-4 and –5.

When FSH is applied, co-dominant follicles become larger and have higher IGFBP-4 and -5 proteolytic activity than subordinate follicles (RIVERA and FORTUNE 2003).

Taken together, these findings suggest that IGFBPs play an important role in follicular selection. FSH induces the proteolysis of IGFBPs, leading to an increase in intrafollicular concentration of free IGF-I that, in turn, synergizes with FSH to promote greater estradiol production by the follicle destined for dominance.