18

present in ovarian

follicular fluid of

sheep. 2. To study the in vivo effects of isolated peptide(s) in rats.

3. To examine the effect of isolated peptide(s) on oocyte maturation, cumulus cell,

oviductal cell and preantral follicle growth in vitro in sheep and buffalo.

4. To examine the effect of isolated peptide(s) on the biochemical profiles in the

cultured oocytes of sheep and buffalo.

2. REVIEW OF LITERATURE

A fundamental understanding of the reproductive cycle of ruminants is essential

for sound reproductive management. The growth and development of follicles and the

oocytes are regulated by changes in the secretion and patterns of different hormones and

local factors (Webb et al., 1997). A better insight into the mechanisms of control of

production of these regulatory substances as well as their interactions during

folliculogenesis, oocyte maturation and ovulation and the interaction of somatic and

gametic compartments of the ovary at their cellular and molecular levels will give a better

understanding of the control of the folliculogenesis and ovulation. Studies on the local

19

nonsteroidal regulators (peptides and polypeptide growth factors) of ovarian functions are

of great interest (Tsafriri and Adashi, 1994) and the results of such studies can be utilized

to develop better strategies for increasing reproductive efficiency not only in domestic

ruminants including cattle, buffaloes, sheep, goats etc. but also in humans (Guraya, 1997).

2.1 Folliculogenesis Folliculogenesis is an ordered sequence of maturation and differentiation steps

involving both somatic and germ cells and culminates in the production of an oocyte

competent to undergo fertilization and subsequent embryonic development (Glister et al.,

2003). It is the formation of mature preovulatory follicles from a pool of primordial

follicles. Various terms had been proposed to describe folliculogenesis (Hodgen, 1982):

a) Recruitment: The entry of an arrested primordial follicle into the pool of growing

follicles is termed recruitment or the primordial-to-primary follicle transition. However,

two stages of follicle recruitment has been reported, first at the primordial and secondly at

the antral stages, when the influence of the gonadotrophins becomes decisive (Webb et

al., 1997), b) Selection: a process whereby a follicle avoids atresia and undergoes further

development and becomes competent to achieve timely ovulation, c) Dominance: a

process whereby a single follicle achieves and maintains its eminence over the other

recruited follicles that undergo atresia.

The fundamental developmental unit of the mammalian ovary is the ovarian follicle.

Ovarian follicles begin their development as primordial structures, which consist of an

oocyte arrested at the dictyate stage of prophase I, surrounded by a few flattened somatic

cells (granulosa cells). The number of oocytes present at birth varied according to species

(ranging from 10000’s in mice to millions in domestic species and humans (Gosden and

Telfer 1987). Once the population of primordial follicles had been established, follicles

were continuously recruited to grow and this growth was independent of the pituitary

gonadotrophins (Girish Kumar et al., 2005). During the growth period the oocyte grew

and granulosa cells proliferated to form a multilaminar structure (preantral follicle).

During the preantral follicle stage, when most oocyte growth occurred, local regulation

appeared to be in operation, involving growth factors such as fibroblast growth factor

(FGF), epidermal growth factor (EGF) and transforming growth factor- (TGF-)

together with 2 proteins (c-kit, present on the oocyte membrane, and its ligand KL,

20

produced by granulosa cells) (Driancourt and Thuel, 1998). Once the follicle reached a

certain size it formed a fluid filled spaces (antrum) with the transition from preantral to

antral follicle.

The stages of follicular development in ewes had been classified (McNatty et al.,

1999) as a) Type 1 (Primordial): follicles with one layer of flattened granulosa cells, b)

Type 1a (Transitory): follicles with one layer of granulosa cells that are a mixture of

flattened and cuboidal granulosa cells, c) Type 2 (Primary): follicles with one to two

layers of cuboidal granulosa cells, d) Type 3 (small preantral)): follicles with two to four

layers of granulosa cells, e) Type 4 (large preantral): follicles with four to six layers of

granulosa cells and f) Type 5 (early antral): follicles with more than five layers of

granulosa cells with evidence of an antrum and g) Preovulatory: large antral follicles to be

ovulated. Follicular development in ewes, from primordial to preovulatory stage,

required about six months, with 2-3 follicles leaving the pool of primordial follicles to

begin growth each day (Cahill and Mauleon, 1980). Three waves of follicle growth and

atresia in a 17-day normal oestrous cycle (two waves during the luteal phase, one wave

during the follicular phase) in ewes were recorded (Noel et al., 1993). No between–

follicle interaction and no negative role of the dominant follicle in differentiation of other

follicles was found (Driancourt, 1994).

Ewes start to show breeding activities when days are shortening (short-day breeders).

The length of breeding seasons differs among the breeds. In general, the hill temperate

breeds had short breeding seasons, whereas lowland temperate breeds have longer

seasons (Gordon, 1997). The oestrous activity in tropical breeds might not have a distinct

period of acyclicity as in the seasonal temperate breeds. No significant seasonal variation

in the length of the oestrous cycle or the duration of oestrus was reported in Rajasthani

breeds (Mittal, 1985). However, breeds of sheep in southern India were reported to be

seasonally anoestrous during March to April and September to October (Kaushish, 1994).

In general, more proportion of ewes exhibited behavioural oestrus during summer than in

winter. The onset of oestrus was relatively earlier during summer than in winter, but the

difference in mean intervals between seasons was not significant. The mean duration of

oestrus was shorter during winter compared to summer (Das et al., 2001).

2.1.1 Intraovarian peptides regulating folliculogenesis

21

Follicular development is controlled partly by intraovarian and intrafollicular

endocrine events within the ovary and partly by extraovarian events (Tsafriri and Adashi,

1994). The growth and differentiation of the ovarian follicle is primarily under the control

of follicle stimulating hormone (FSH) and luteinizing hormone (LH) secreted by pituitary

glands (sheep: Campbell et al., 1999a; cattle: Mihm and Bleach, 2003). However, the

recent studies indicated that folliculogenesis was modulated by various growth factors,

novel proteins (Armstrong and Webb, 1997) and other local factors which include

aromatase inhibitor (Dizerega et al., 1982), follicle regulatory protein (Tonetta et al.,

1986), anti-mullerian hormone (AMH, also called Mullerian inhibitory substance, MIS)

(Bezard et al., 1987), oocyte maturation inhibitor (OMI, a polypeptide of 1.5 kDa, Sirard

and First, 1988), inhibin (FSH suppressing factor, Burger, 1988), activin or FSH releasing

protein, follistatin (Findlay, 1993), FSH binding inhibitor, luteinizing stimulator,

luteinizing inhibitor, steroid binding proteins, enzymes (plasminogen, proteases),

fibronectin inhibitory protein (a polypeptide of 14 kDa), gonadotrophin releasing

hormone (GnRH), GnRH-like proteins (Guraya, 1997), growth differentiation factor-9

(GDF-9), bone morphogenetic protein (BMP-15), pregnancy-associated plasma protein-A

(PAPP-A) (Erickson and Shimasaki, 2001), inhibin binding protein (InhBP, Bernard and

Woodruf, 2001). All the intraovarian peptides except inhibin appeared to act locally

within the follicle/ovary whereas the inhibin activity in follicular fluid was determined on

the basis of extra-ovarian actions, which consist of selective inhibition of FSH secretion

in vivo (Guraya, 1997). Inhibin/activin/follistatin and growth factors in the ovarian

follicular fluid were studied in depth to use them for regulation of fertility in humans and

domestic animals.

2.1.1.1 Inhibin/Activin/Follistatin Inhibins and activins are dimeric glycoproteins consisting of and subunits, and

follistatin is a high affinity activin binding monomeric glycoproteins. Follistatin (35 kDa

peptide) was originally identified as an inhibitor of FSH secretion by cultured pituitary

cells, but its potency was only 10-30% of that of inhibin (Ueno et al., 1987). Dimeric

inhibins in follicular fluid are high molecular weight (>160 kDa) forms while smallest

dimeric forms (32-34 kDa) are in low levels (Austin et al., 2001). Gonadal inhibins

reported to function as negative feedback hormones to regulate the synthesis and

22

secretion of pituitary FSH, a key determinant of follicle development, but there was little

supportive evidence for a peripheral endocrine role for ovary-derived activins or

follistatin in this regard (Knight and Glister, 2001). Inhibin content in ewes was

significantly higher, in large antral follicles compared to small and medium-sized

follicles, however, no significant increase was observed between medium and small

follicles (Shidaifat, 2001). Activin (a 25 kDa peptide, Grootenhuis et al., 1989) induced

granulosa cell proliferation; increased FSH receptor expression; granulosa cell

steroidogenesis, basal and gonadotrophin stimulated aromatase activity, estradiol

production and delayed the onset of lutenization and atresia (Knight and Glister, 2001,

Beg, 2005). The intra-follicular ratios of activin: follistatin and activin: inhibin had been

suggested to be potentially important parameters regulating folliculogenesis. Follistatin

decreased the activity of activin by binding it and inhibin opposes the actions of activin.

Thus a ratio of activin: follistatin and activin: inhibin reflected the net amount of

unopposed activin (activin tone) likely available for interaction with its receptors (Beg,

2005).

2.1.1.2 Growth factors Growth factors are proteins (

23

I was involved in the increase in FSH-responsiveness of granulosa cells when follicles

enter into the gonadotrophin-dependent stages of follicular development (200 m in

mouse, 2 mm in sheep, 5 mm diameter in cattle) (Monget and Bondy, 2000). In vitro

effects of IGF-I in bovine include increased granulosa cell proliferation and estradiol

production, enhanced sensitivity of granulosa cells to FSH, increased secretion of inhibin,

activin and follistatin from theca cells and enhanced LH stimulated androgen synthesis

from theca cells (Glister et al., 2003). The IGF-I enhanced in vitro maturation of oocytes

in cattle (Iga et al., 1998), sheep (Guler et al., 2000) and buffalo (Arthi, 2003), while

IGF-II increased maturation of bubaline oocytes in vitro (Chauhan et al., 1998).

Actions of IGF-I and -II are restrained by a series of IGF binding proteins (IGFBPs)

that either originate from the blood or are synthesized locally within the follicle. Various

forms of IGFBPs were reported with different molecular weight, amino acid composition,

binding properties, distribution in biological fluids and influence upon IGF activity

(Kostecka and Blahovec, 1999). During follicular growth, levels of IGFBP-2, -4 and -5

decreased in follicular fluid. They led to an increase in the bioavailability of IGFs and

their action on granulosa cells. By contrast, follicular atresia was characterized by a high

increase in the synthesis and levels of IGFBPs and a decrease in IGFs bioavailability. In

sheep, IGF-I stimulated granulosa cells from small follicles to proliferate and those from

larger follicles to produce progesterone, an effect likely mediated through the type I IGF

receptor (Poretsky et al., 1999). IGFBP proteases had been recently described in bovine

follicular fluids (Spicer et al., 2004). The proteases degraded the binding proteins and

thus increase the bioavailability of IGFs in the follicle (Beg, 2005).

2.1.1.2.2 Epidermal growth factor (EGF)

The EGF family comprised EGF, transforming growth factor -, heparin-binding

EGF, epiregulin, amphiregulin, betacellulin, epigen and neuregulins (Werner and Grose,

2002). EGF is a single chain polypeptide of 53 amino acids with molecular weight of 6

KDa (Raaberg et al., 1990). EGF was reported to regulate follicle cell proliferation,

protein synthesis and the production of progesterone and inhibin by granulosa cells. EGF

promoted the proliferation of granulosa cells from preantral and antral follicles, decreased

the production of inhibin and progesterone while stimulating the DNA and protein

synthesis and suppressed the FSH-induced induction of LH receptors in granulosa cells

24

(Guraya, 1997). Nuclear maturation of cattle oocytes was not affected when denuded

oocytes were cultured in media containing EGF suggesting a link between the EGF and

cumulus cell (Bevers et al., 1997) EGF also enhanced the in vitro maturation of cattle

(Goff et al., 2001) and buffalo (Nandi et al., 2003a) oocytes.

2.1.1.2.3 Fibroblast growth factor (FGF) Fibroblast growth factor family consisting of 22 members (Ornitz and Itoh, 2001) had

been isolated in both acidic (pI 4.5, aFGF) and basic (pI 9.6, bFGF)) forms, each being of

Mr 16-17Kda (Gospodarowicz et al., 1987). FGF was potent mitogen for tissues derived

from mesenchyme or neuroectoderm such as chondrocytes, fibroblasts, myoblasts, glial

cells, adrenal cortex and ovarian granulosa cells. Basic FGF produced by granulosa cells

regulate their differentiation and invasion by capillaries. On one hand, FGF promoted

follicular development and corpus luteum formation and on the other, by virtue on its

action on aromatase activity, it could inhibit estradiol formation and effectively mediate

follicular atresia (Baird et al, 1986). In cattle, no significant effect was achieved with FGF

on cumulus expansion of oocytes (Kobayashi et al. 1994). However, Bieser et al. (1998)

reported a finely tuned extracellular proteolysis during in vitro maturation of oocytes, for

which the action of modulating growth factor like FGF was essential. In buffalo, FGF

stimulated the oocyte maturation in vitro, however, its role in less than that of EGF

(Nandi et al., 2003a).

2.1.1.2.4 Transforming growth factor (TGF)

TGF comprised of TGF- (7.5 KDa), TGF-1 (25 kDa) and TGF-2 (25 kDa)

(Roberts et al., 1988). TGF- differed from other growth factor as it acted as inhibitor of

cell proliferation (Carson et al., 1989). The TGF--related family of proteins included the

activin and inhibin proteins, MIS, BMP and other as many as 100 distinct proteins. TGF-

(an EGF-like growth factor) was a mitogenic agent secreted by tumor cells, binds to

EGF receptors, and causes similar effects (Guraya, 1997). The role of TGF in bovine

oocyte in vitro maturation was controversial; a negative role of TGF- was reported by de

Loos et al., 1992, while Kobayashi et al., (1994) demonstrated TGF- and TGF-

increased cumulus expansion and nuclear maturation.

25

2.1.1.2.5 Platelet-Derived Growth Factor (PDGF) PDGF comprised a family of homo-and heterodimeric growth factors, including

PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD with molecular weight

ranging from 28 to 31 kDa (Heldin et al., 2002). Purified PDGF was shown to enhance

FSH-induced progesterone secretion by granulosa cells, adenylate cyclase activity and

LH receptor induction (Carson et al., 1989). PDGF stimulated the in vitro maturation of

cattle oocytes (Eckert and Niemann, 1996).

2.1.1.2.6 Tumor Necrosis Factor (TNF)

Two forms of Tumor Necrosis Factors (TNF) were present namely TNF- and

TNF-. TNF- (also called cachectin) was a major immune response modifying cytokine

produced primarily by activated macrophages. TNF-α inhibited gonadotrophin supported-

progesterone accumulation by differentiating and luteinized murine granulosa cells

(Adashi et al., 1990). TNF- (also called lymphotoxin) was characterized by its ability to

kill a number of different cell types, as well as the ability to induce terminal

differentiation in others. Potential role of TNF-α on the ovarian cells of rat, rabbit, guinea

pig, cattle, pig and women was reviewed by Terranova (1997). TNF-α was involved in

follicle development, ovulation, luteal development and regression. In small developing

follicle, TNF-α suppressed the responsiveness of the ovary to gonadotrophins, whereas in

preovulatory follicles it stimulated steroidogenesis. TNF-α secreted by dominant follicles

had a paracrine role in suppressing the development of smaller follicles (Terranova,

1997). TNF-α also regulated collagenolytic activity in preovulatory ovine follicles

(Johnson et al., 1999).

2.1.1.2.7 Hepatocyte Growth Factor (HGF) A single chain precursor (Mr -87 KDa) cleaved to 69 and 34 kDa chain that was

linked with one disulfide bond to make HGF (Parrott and Skimmer, 1998). Both single

chain and cleaved forms were active. HGF stimulated divisions in hepatocytes, renal

tubular epithelial cells, epithelial keratinocytes, melanocytes and epithelial granulosa

26

cells. HGF was produced by theca cells in the ovary and stimulated the granulosa cell

proliferation during follicular development (Parrott and Skimmer, 1998).

2.1.1.2.8 Nerve Growth Factor (NGF) Two identical subunits (13.6 kDa) formed NGF. NGF was responsible for

development and maturation of sensory and sympathetic neurons (Werner and Grose,

2002). GnRH caused a rise of NGF in large ovarian follicles in cattle (Guraya, 1997). The

increase in NGF secretion following gonadotrophin surge suggested that NGF was

involved in the control of oocyte maturation. Sheep follicles also produced NGF in the

presence of suitable hormonal milieu (gonadotrophin surge). Under these conditions the

production of the NGF increased with increasing follicle size (Mattioli et al., 1999). In in-

vitro conditions, NGF (100 ng/ml) induced a marked cumulus expansion and a

progressive cumulus-oocyte uncoupling similar to that produced by gonadotrophins in

sheep. The addition of NGF also caused the resumption of meiosis in more than 70% of

the oocytes analyzed with an effect that was only slightly less pronounced than that of

gonadotrophins (80%) (Barboni et al., 2002).

2.1.1.2.9 Vascular Endothelial Growth Factor (VEGF) The VEGF family included VEGF-A, VEGF- B, VEGF-C, VEGF-D, VEGF-E, and

placenta growth factor (Werner and Grose, 2002). The level of VEGF, an angiogenic

factor, increased as the follicle diameter increased (Berisha et al., 2000). VEGF had

shown to stimulate the mitosis of endothelial cells, increased vascular permeability and

angiogenesis. VEGF system had an angiogenetic effect during in vivo and in vitro

maturation of the bovine oocytes, possibly affecting the early embryonic viability

(Einspanier et al., 2002). VEGF production increased in cultured granulosa cells in cattle.

Follicle produced VEGF also had a role in vascular follicle relationships during diameter

deviation (Beg, 2005).

2.1.1.2.10 Vasoactive intestinal peptide (VIP) VIP was present in the ovarian stroma of mammals and form association with

antral follicles in immature rat ovaries (Guraya, 1997). VIP stimulated the synthesis of

27

progesterone, 20- hydroxyprogesterone and oestrogen (Guraya, 1997). Culture of

bovine oocyte in the presence of VIP did not affect nuclear maturation or cumulus

expansion, but it retarded cytoplasmic maturation (Beker et al., 2000). Addition of VIP to

the culture medium did not improve in vitro maturation of sheep (Ledda, 1996) and

buffalo oocytes (Nandi et al., 2003a)

2.1.1.2.11 Cytokines Little information was available on the role of cytokines in follicle growth and

development. IL- was more potent than IL- in suppressing FSH-induced differentiation

of granulosa cells. The participation of macrophages in proliferation of granulosa cells as

local mediators in growing follicles (Fukumatsu et al., 1992) could be attributed to some

growth factors secreted by macrophages. The expression of IL-1β gene in granulosa cells

and the follicular fluid IL-1β content seemed to be regulated by gonadotrophins

suggesting that IL-1β could be an intermediate paracrine factor involved in ovulation

(Martoriati and Gerard, 2003).

The availability of modern molecular biology techniques like suppressive

subtraction hybridization and gene array techniques had made it possible to screen and

identify genes that are differentially expressed in dominant and subordinate follicles. It

had been reported that dominant follicles had enhanced expression of antiapoptotic tumor

associated genes (DICE-I, MLC-I) whereas the subordinate follicles had higher

expression of proapoptotic genes like TNF-, DRAKE-2, CAD, COX-I in granulosa and

bGlycan, Apaf-I, BTG-3, caspase-13 in the thecal cells (Beg, 2005).

2.2. Follicular Fluid The oocyte and granulosa cells in the ovary were bathed in a fraction of

extracellular fluid called follicular fluid, which accumulated in the antrum of ovarian

follicles. Follicular fluid was the in-vivo environment of the oocyte during its maturation.

It contained numerous biochemical components that are essential for ovarian physiology,

including steroidogenesis, follicle growth and ultimate maturation of oocytes, ovulation

and oviductal transport of the oocyte (Edwards, 1974). Follicular fluid was in part

exudates of serum, as surrounding cell layers permit the free diffusion of proteins of up to

28

500 kDa (Payer, 1975). It was a complex of restricted components of serum and follicular

synthesized secretions (Wise, 1987).

Biochemical Composition of follicular fluid One of the factors controlling ovarian physiology in animals was the alteration

that occurred in the biochemical composition of follicular fluid. The dynamic constituents

of follicular fluid reflected both biochemical and endocrinological activity of the follicle,

thereby facilitating its role as a conductor of growth and development through the

reproductive cycle (Edwards, 1974). Major important components of follicular fluid are

the gonadotrophins and steroid hormones. The changes of levels of gonadotrophin and

steroids in ovine follicular fluids during follicular development were reported in exotic

McNatty et al., (1981) and Indian (Shailaja et al., 1985) breeds. The hormonal

composition of follicular fluid in ewes and its variation due to nutrition and

superovulation was reported by O'-Callaghan et al., (2000). Though the blood

biochemical (non-hormonal) profile in Indian sheep breeds was available (Jagatheesan et

al., 2003), the information on non-hormonal composition of follicular fluid of ewes, both

exotic and Indian breeds was ill defined.

2.2.1.2. Glucose, protein and cholesterol content 2.2.1.2.1. Glucose Cattle: Biochemical analysis revealed that glucose was ~75% in the total carbohydrate

contents of bovine follicular fluid with fructose being present as only a minor fraction

(Lutwak-Mann, 1954). An increase in glucose level was observed in the follicular fluid

from small (2.010.10mM), medium (2.850.16mM) to large (3.750.18mM) follicles

(Leroy et al., 2004). The glucose level in cystic follicles was reported to be lower than

that of preovulatory follicles (Boryczko et al., 1995).

Buffalo: The concentration of glucose increased markedly during the early stages of the

cycle (Eissa, 1995). Follicular fluid contained lower concentrations of glucose compared

with blood plasma (Ahmed et al., 1997, Jindal et al., 1997).

Goat: The mean glucose concentration was non-significantly higher in large follicles

compared to small ones (26.31±2.15 vs. 19.44±2.18 mg%) (Thakur et al., 2003).

29

Pig: No difference in the glucose concentration of follicular fluid was observed among

various sized follicles. The average glucose concentrations were 86, 86 and 83 mg/dl in

follicular fluids of large, medium and small follicles, respectively (Huang et al., 2002).

2.2.1.2.2. Protein Cattle: Protein concentrations had been reported to be ~75% of that of serum (Edwards,

1974). Analysis of proteins in follicular fluid provided evidence that follicular fluid is in

partial equilibrium with serum in sheep, pigs and cows (Cook et al., 1977). Total protein

concentration decreased as follicular diameter increased (Wise, 1987). The total protein

was found to be 6.59± 0.10, 6.36±0.11 and 6.50±0.10 g/dl in small, medium and large

follicles, respectively (Leroy et al., 2004).

Buffalo: The concentration of total protein increased markedly during the early stages of

the estrous cycle (Eissa, 1995). Follicular fluid contained lower concentration of total

protein compared with blood plasma (Ahmed et al., 1997, Jindal et al., 1997). A declining

trend of protein concentration from small to large follicles (Small: 6.50.19, medium:

6.480.22 and large: 6.280.23 g/dl) was observed (Thangavel and Nayeem, 2004). The

total protein concentration was higher in double ovulated (6.5 g/dl) than cystic (3.9 g/dl)

or normal (3.4 g/dl) ovaries, respectively (Zeitoun, 2002). The total protein concentration

in the small, medium and large follicles of the left ovaries were 6.9, 6.25 and 6.6 g%,

respectively while the corresponding values for the right ovaries were 7.2, 7.0 and 7.0

g%, respectively (Mohan et al., 1997).

Sheep: The total protein concentration in follicular fluid of small, medium and large

follicles were found to be 6.86, 6.05 and 5.71 g/dl, respectively (Balakrishna, 1994). In

contrast, protein concentration was found to increase as follicular diameter increased

(Singh et al., 1999).

Goat: Large follicles had lower total protein (3.76±0.66 vs. 4.8±0.44 g%) compared to

small follicles (Thakur et al., 2003). The concentration of protein decreased significantly

from small (5.800.36 g/dl) to medium (3.970.12 g/dl) and from medium to large

(2.960.14 g/dl) follicles (Mishra et al., 2003).

Pig: The protein concentration was found to decrease as the follicle size decrease. The

average protein concentration was 73.97±9.47, 56.82±7.19 and 59.87±6.86 mg/dl in

large, medium and small follicles, respectively (Huang et al., 2002).

30

An increase in total protein concentration in follicular fluid with increase in size

of follicle was reported in cattle (Brantmeier et al., 1987), sheep and goats (Singh et al.,

1999) and buffalo (Thangavel and Nayeem, 2004). A decrease in the total protein

concentration was observed in cattle (Wise, 1987) and sheep (Balakrishna, 1994). In

contrast, no difference in the total protein concentration in follicular fluid was observed

among various sized follicles in goats (Sidhu et al., 1985), buffalo (Parmar and Mehta,

1991b) and cattle (Leroy et al., 2004).

2.2.1.2.2. Cholesterol Cattle: The total cholesterol was found to be 55.9±3.39, 62.7±2.91 and 63.7±3.23 mg/dl

in small, medium and large follicles, respectively (Leroy et al., 2004).

Buffalo: The cholesterol concentration was reported to be significantly higher in medium

sized follicles (80.773.96mg%) than in small (74.933.52mg%) and large

(74.903.39mg%) follicles (Thangavel and Nayeem, 2004).

Goat: The mean concentration of cholesterol in the follicular fluid was significantly

higher in the large compared with the small follicles (61.50±1.03 vs. 54.40±1.38 mg%)

(Thakur et al., 2003). Similar observation was reported by Bordoloi et al., (2000). The

concentration of cholesterol in the follicular fluid indicated a significant increase from

small (43.673.42 mg/dl) to medium (70.1515.35 mg/dl) and to large (172.2814.34

mg/dl) follicle (Mishra et al., 2003).

Pig: No difference in the cholesterol concentration in follicular fluid was observed among

various sized follicles. The average cholesterol concentrations were 153, 161 and 157

mg/dl in large, medium and small follicles (Huang et al., 2002).

2.2.1.3. Inorganic components Cattle: Sodium concentration in follicular fluid was greater in growing-antral than atretic

follicles, and the concentration increased with follicular enlargement (Wise, 1987).

Follicular potassium concentration increased as the oestrous cycle progressed, and tended

to be elevated in atretic follicles. Both calcium and magnesium concentrations increased

with follicular enlargement (Wise, 1987). A decrease in the concentration of potassium in

follicular fluid from small to large ovarian follicles was observed. No significant variation

in the sodium concentration in follicular fluid in various sizes of follicles was also

31

reported. The sodium and potassium concentrations of follicular fluid were reported to be

142.5±0.34, 142.4±0.63 and 145.0±0.64; and 10.1±0.21, 7.9±0.28 and 6.0±0.23 mM,

respectively in small, medium and large sized follicle (Leroy et al., 2004).

Buffalo: The concentrations of calcium and phosphorus increased markedly during the

early stages of the cycle (Eissa, 1995). Calcium concentration was higher in large follicles

(8 ± 1.2 µg/ml) than in small (6.9 ± 1.1 µg/ml) and medium follicles (5 ± 1.6 µg/ml)

(Kaur et al., 1997). The concentrations of magnesium and potassium were significantly

higher in ovarian tissues than in follicular fluid. The levels of magnesium and calcium in

the ovaries of cyclic buffaloes were 46.4±3.64 and 16.0±0.73 µg/ml, respectively. Their

levels in the ovaries of pregnant animals were 52.0±3.13 and 17.4±1.33 µg/ml,

respectively. No significant difference in the levels of these macro- and microminerals

were observed in the ovarian tissues of cyclic and pregnant buffalo (Bhardwaj et al.,

1998). Concentration of magnesium was significantly lower in large follicles (10.4±2.5

µg/ml respectively) than in small (25±5.8 µg/ml) and medium (26.9±4.9 µg/ml) follicles

(Kaur et al., 1997). They also observed that the concentration of sodium was significantly

higher in fluid from large follicles (75±4.2 µg/ml) than in fluid from small and medium

follicles (25±2.4 and 25±2.9 µg/ml). Sodium and calcium concentrations increased while

potassium and magnesium concentrations decreased with the follicular development

(Kalmath, 2000)

Sheep: The sodium, potassium, chloride, calcium and magnesium concentrations in

overall follicular fluid was found to be 149, 4.7, 107, 2.29 and 0,89 mmol/ml (Gosden et

al., 1988).

Goat: The concentrations of calcium and magnesium increased significantly in large

follicles, but that of phosphorous decreased significantly as the size of the follicle

increased (Bordoloi et al., 2001). It was observed that the potassium concentration

decreased significantly as the follicles increased in size (Bordoloi et al., 2001). Large

follicles had lower potassium (13.0±0.61 vs. 16.25±1.30 mEq/litre) compared to small

follicles. Magnesium and sodium were higher in the large follicles; however, the

differences were not significant (3.05±0.26 vs. 2.50±0.22 mEq/l and 97.25±9.69 vs.

84.00±0.58 mEq/l, Thakur et al., 2003). The concentration of inorganic phosphorus did

not reveal any significant difference between the different size follicles (Mishra et al.,

2003). The sodium concentration in ovarian follicular fluid varied significantly among

32

different sizes of follicles. The mean sodium concentration in follicular fluid of large

follicle (134±1.18 mEq/l) was significantly higher than small (115.67±1.9 mEq/l) and

medium (119.67±3.48 mEq/l) follicles.

Pig: Sodium, potassium and calcium content decreased with follicular development

(Schuetz and Annisowicz, 1974). The potassium concentration was higher in small (days

12-13) and medium-sized (day 16) follicles than in large (day 18, oestrus) follicles or in

the plasma of cyclic sow. In contrast, follicular fluid obtained from slaughterhouse

material had a higher potassium concentration and osmolality, and a lower sodium

concentration than those in the plasma or follicular fluid of the cycling pig, and these

differences are assumed to be due to post-mortem changes (Knudsen et al., 1979).

2.2.1.4. Enzymes Cattle: As follicular diameter increased, acid phosphatase, alkaline phosphatase and

lactate dehydrogenase activity in follicular fluid was reduced (Wise, 1987). Alkaline

phosphatase and acid phosphatase fluctuated, and the level of lactate dehydrogenase

declined with follicle growth. There were significant differences between the activity of

an enzyme in ovaries with and without corpora lutea (Dave, 1981). Total acid

phosphatase activity per follicle increased with follicle size. Alkaline phosphatase activity

was greater in the smallest follicles (Henderson and Cupps, 1990).

Buffalo: Phosphatase activity tended to decrease as follicle size increased (Eissa, 1995;

Kalmath, 2000). No significant difference in acid phosphatase and alkaline phosphatase

activity among follicles of different size was also reported (Parmar and Mehta, 1991a).

The levels of alkaline phosphatase were 783.0, 147.2 and 112.5 IU/litre, acid phosphatase

values were 82.1, 1.2 and 2.6 IU/litre, for double-corpus luteum, cystic and normal

buffalo ovaries respectively (Zeitoun, 2002).

Sheep: Acid phosphatase in follicular fluid of 3-6 mm follicles showed no significant

modifications between healthy and initially atretic follicles and only a small but

significant increase in activity in advanced atretic follicles (Rosales et al., 2000). Goat: Acid phosphatase increased progressively from small (17.682.09 KAU/dl) to

medium (23.991.61 KAU/dl) and from medium to large (28.090.82 KAU/dl) follicles.

Alkaline phosphatase activity showed reverse trend (small: 37.475.8 KAU/dl), medium

33

(31.422.92 KAU/dl) and large (24.781.12 KAU/dl, Mishra et al., 2003). Though

follicular fluid acid phosphatase activity was higher in small than in medium and large

follicles, no significant difference of alkaline phosphatase activity was observed between

the classes of ovarian follicle (Bordoloi et al., 1999).

Pig: A progressive decrease in acid and alkaline phosphatase activity with follicular

development was reported (Chang et al., 1976).

The concentrations of various components of follicular fluid varied during

different stages of the reproductive cycle, seasons, environmental temperature, nutritional

status of the animal and due to different ways of estimation in the various laboratories.

Concentrations might vary widely between different follicles in the same animal or

among animals of the same species and different species.

2.2.2. Electrophoretic analysis of follicular fluid proteins Cattle: Forty proteins were detected in follicular fluid studied by gel chromatography and

quantitative immunoelectrophoresis (Andersen et al., 1976). Serum and follicular fluid

compared by electrophoresis revealed that serum showed both alpha- and beta-

lipoproteins, while beta-lipoprotein was almost totally absent from follicular fluid

(Vukotic et al., 1979). Follicular fluids examined by polyacrylamide-gel electrophoresis

showed that the alpha -globulin 3 from large follicles migrated faster than the -globulin

3 from small follicles (Segerson and Gray, 1978). Three major zona pellucida proteins

(ZP1, ZP2 and ZP3), with apparent molecular weights of 80-70, 66-63 and 60 kDa

respectively were revealed by one-dimensional sodium dodecyl sulphate polyacrylamide

gel electrophoresis (SDS-PAGE). Two-dimensional SDS-PAGE indicated the presence of

a 4th glycoprotein (ZP4) (Bercegeay et al., 1993). Follicles > 10 mm in diameter

contained a polypeptide with a molecular weight between 39 and 43 KDa and was similar

to IGF-binding protein-3 (IGFBP-3), which was not observed in follicles < 5 mm and 5-

10 mm in diameter (Gradela et al., 1998). Matrix metalloproteinase-2, a protein of 110-

kDa having gelatinolytic activity was demonstrated in follicular fluid (Minjung et al.,

2001). Two-dimensional polyacrylamide gel electrophoresis map of bovine ovarian fluid

proteins was examined (Mortarino, et al., 1999) and a quantitative difference in the

protein pattern of follicular fluid with follicular development was reported.

34

Buffalo: SDS-PAGE of follicular fluid revealed 26 protein bands (Kulkarni, 1988) and

was identical to that of blood plasma proteins. No differences were observed in the SDS-

PAGE spectra of small, medium and large follicles. Mohan et al., (1997) examined the

electrophoretic pattern of the albumin and globulin of follicular fluid and reported that the

globular fraction was higher in small follicles. Muralikrishnan (2002) reported 37 bands

in buffalo follicular fluid, irrespective of the size of follicles.

Sheep: Polypeptides of 46 and 60-kDa secreted by granulosa and cumulus cells in the

ovine follicular fluid before exposure to the preovulatory LH surge were reported by

Moor and Crosby, (1987). IGF-binding proteins (IGFBP) were identified by 12% sodium

dodecyl sulphate polyacrylamide gel electrophoresis in follicular fluid (Khalid and

Haresign, 1996). The intensity of the IGFBP at 44-42 kDa was significantly higher in

follicular fluid from oestrogenic follicles, whereas the intensity of the band at 35 kDa was

significantly higher in follicular fluid from non-oestrogenic follicles. Some of the non-

oestrogenic follicles also exhibited bands at 32.0-28.5 kDa with variable intensities, but

such bands were totally absent in oestrogenic follicles. Proteins secreted by sheep ovarian

follicles at different stages of maturation (small healthy, large healthy or large atretic) and

originating from ewes with different ovulation rates (homozygous carriers or non-carriers

of the FecB gene) were analyzed by 2-dimensional electrophoresis (Driancourt et al.,

1996a, b). The presence of the FecB gene induced qualitative and quantitative changes in

the follicle protein patterns. Size and atresia of follicle affected protein patterns only

quantitatively. Follicular fluid and serum proteins were analyzed between high and low

ovulation rates in Finn ewes using 2D polyacrylamide gel electrophoresis (PAGE). A line

difference was detected for a serum protein (40 kDa). High-line ewes had 1 spot, whereas

low-line ewes had 3 spots on 2D PAGE. The effect of the Booroola gene (FecB) on

oocyte characters was studied in FecBFecB, FecBFec+ and Fec+Fec+ ewes (Cognie et

al., 1998). Resolution of the proteins synthesized by FecBBB and Fec+Fec+ oocytes by

one-dimensional polyacrylamide gel electrophoresis and image analysis demonstrated

quantitative (but not qualitative) differences between genotypes for bands at 74, 59, 35

and 25 kDa. A genotype X oocyte size interaction was detected for 2 additional bands at

45 and 43 kDa.

Goat: Electrophoresis of the blood serum in goats revealed 6 bands, one of which, the

r-globulin band, was absent from the follicular fluid. Another band, present in the

35

follicular fluid but not in the serum, was -globulin synthesized within the follicle

(Sarmah et al., 1998).

Horse: A 200 Kda protein band was reported in preovulatory follicles of mare, which

was absent in the follicles in earlier stage, subordinate follicles and serum (Gerard et al.,

1998).

Human: Human follicular fluids were examined by two-dimensional gel electrophoresis

and 60 proteins were detected (Spitzer et al., 1996). Follicular fluid samples were also

processed for high-resolution two-dimensional polyacrylamide gel electrophoresis (2-D

PAGE) (Anahory et al., 2002). Comparative analysis of the 2-D gels revealed up to 600

spots, of which four were selected because of variations in their expression level. Using

direct sequencing procedures (Edman degradation) or matrix assisted laser

desorption/ionization-mass spectrometry (MALDI-MS), these four spots were identified

as three new proteins: thioredoxin peroxydase 1 (TDPX1), transthyretin (TTR) and

retinol-binding protein (RBP).

2.3. Isolation of follicular fluid proteins Majority of the earlier works in follicular fluid protein isolation were carried out in

cattle and porcine species. The most widely studied isolated proteins were inhibins,

oocyte maturation inhibitory factors and follicle growth suppressors.

Cattle: FSH inhibitor (Mr:17kDa) was purified from follicular fluid by precipitating

follicular fluid with ammonium sulphate at a concentration of 14.5 to 18.5%, and the

reconstituted material separated into 2 peaks by Sephadex G-200 column

chromatography. The 2nd peak, detectable as a single band by polyacrylamide gel disc

electrophoresis, contained all the inhibitory activity (Sato et al., 1982). Three FSH-

suppressing proteins (of molecular weight 31, 35 and 39-kDa) with inhibin-like activity

were isolated from follicular fluid by gel filtration (Robertson et al., 1987). A 65-kDa

inhibin from cattle follicular fluid was purified by chromatography and electrophoresis

(Boyhan et al., 1988). The isolation of inhibin alpha-subunit precursor proteins from

follicular fluid was reported by Robertson et al., (1989). Biologically active inhibin was

isolated from follicular fluid by gel permeation chromatography, anion exchange

chromatography and reversed-phase high performance liquid chromatography (Knight et

36

al., 1990). The properties of an immunosuppressive fraction of follicular fluid were

studied after separating the fraction from proteinase inhibitor by column chromatography

(Veselsky et al., 1991). Isolation of nine different biologically and immunologically

active molecular variants of follicular inhibin was demonstrated by Good et al., (1995).

The isolation and purification of the activin/follistatin complex from bovine follicular

fluid by gel filtration were reported by Hasegawa et al., (1996). The protein fraction

responsible for the inhibition of maturation of cattle oocytes in vitro was isolated from

follicular fluid by means of column chromatography on Sephadex G-200 and Sepharose

4B, both in 0.1 M ammonium acetate, pH 6.7. The molecular weight of the maturation-

inhibiting protein fraction (oocyte maturation inhibitor, OMI) was approximately 60 kDa

(Dostal et al., 1996). Low molecular mass factors from cattle follicular fluid having

inhibitory effect on steroidogenesis in bovine granulosa cells was purified by filtration

through membranes in 25 and 10 kDa molecular weight cutoffs (Baratta et al., 2001). An

8.6-kDa protein with sperm-attracting activity was isolated by using size exclusion

chromatography and SDS-PAGE techniques (Serrano et al., 2001). The isolation and

characterization of vascular smooth muscle cell growth promoting factor from follicular

fluid and its cDNA cloning was reported (Miyamoto et al., 2001). A haptoglobin-like

protein (40-kDa) was isolated from follicular fluid of antral follicles by electroelution

from SDS-PAGE gels (Lavery et al., 2003). A novel apolipoprotein (12-kDa protein),

designated ApoN, had been isolated from follicular fluid using chromatographic

purification methods (O’Bryan et al., 2004).

Pig: A 10-55% saturated ammonium sulfate fraction of pooled porcine follicular fluid

(PFF) was dialyzed against 0.025 M Tris/HCl, pH 7.5, using 10 kDa molecular weight

exclusion membranes, then passed through agarose-immobilized textile dye. When

aliquots of the saturated ammonium sulfate precipitated, dialyzed, orange A-bound,

Sephadex G-100 (Ve/Vo 1.3-1.7) eluent were separated by high-performance liquid

chromatography (HPLC) using gel exclusion columns, activity in the bioassay was

recovered in the 18-35 kDa molecular weight range (Kling et al., 1984). Hypoxanthine,

the inhibitor of oocyte maturation was isolated from a low molecular weight fraction of

follicular fluid (Downs et al., 1985). The stimulatory action of follicular fluid components

on maturation of granulosa cells from small follicles was reported by Kolena and

Channing, (1985). After partial purification of pooled fluid from large follicles, or

37

proteins precipitated with 90% ethanol, on Sephadex G-100, the greatest stimulatory

activity was found in the 2nd protein peak eluted from the column. Chromatography of

part of the active fraction yielded 7 protein fractions. The 2nd fraction, which eluted

early, contained most of the stimulatory activity, which was about 32-fold higher than in

untreated fluid (Kolena and Channing, 1985). A low molecular weight substance (1-3

kDa) capable of inhibiting oestradiol and progesterone biosyntheses in rat ovarian

follicles was extracted from small follicular fluid (Kigawa et al., 1986). Follicle

regulatory protein, was purified about 6666-fold from the orange A-bound fraction of

porcine follicular fluid. It inhibited granulosa cell aromatase activity by 50% when added

at a concentration of 25 ng/ml. The inhibitor had a size of 310 kDa and was composed of

3 subunits of 178, 101 and 55 kDa (Ono et al., 1986). An FSH-releasing protein (Mr: 28-

kDa) was purified from follicular fluid having high stimulatory action on the in vitro

secretion and biosynthesis of FSH (vale et al., 1986). An inhibitory polypeptide (Mr: 53-

kDa) that blocked FSH-induced estradiol and progesterone production in rat ovary

granulosa cells was isolated from follicular fluid (Shimasaki et al., 1990). The protein, of

molecular weight 52 kDa having N-terminal sequence similar to that of antithrombin III

was isolated from porcine follicular fluid (Lee et al., 1992). The partially purified low molecular weight luteinizing hormone binding inhibitor in porcine follicular fluids was

obtained by ultrafiltration, gel filtration and anion exchange chromatography. Fractions of

molecular weight 0.5-10 kDa inhibited binding of human LH to pig granulosa cells in a

dose-dependent manner (Kokawa et al., 1993). The purification of high-molecular-weight

follicle-stimulating hormone binding inhibitor in follicular fluids was reported (Furukawa

et al., 1994). A protein of 87 kDa was partially purified from follicular fluid by gel

filtration chromatography (Ramsoondar et al., 1995). The purification of gonadotropin

surge inhibiting factor from follicular fluid was reported by Danforth and Cheng, (1995).

The factor(s) in follicular fluid arresting the induction of cumulus expansion of oocyte-

cumulus complexes was purified by Daen et al., (1997). The porcine follicular fluid was

fractionated by ultracentrifugation (220 000 g for 48 h) to give 4 fractions. Cumulus-

oocyte complexes cultured in the top fraction expanded more than complexes cultured in

the other fractions. When the bottom and top fractions were mixed, there was a decrease

in the degree of expansion of the cumulus oophorus in vitro. When oocyte complexes

were re-cultured in the top fraction for a further 24 h, expansion of the cumulus oophorus

38

resumed. The bottom fraction was dialysed with molecular cutoff points of 10, 25 and 50

kDa. The inhibitory activity was retained after dialysis with the molecular cut off point of

50 kDa (Daen et al., 1997). Follipsin complexed with 2-macroglobulin was

demonstrated and characterized from ovarian follicular fluid (Kohyama et al., 1998).

Buffalo: Buffalo follicular fluid separated into 2 fractions when subjected to column

chromatography after treatment with ammonium sulphate. Both fractions possessed the

inhibitory activity (Kumar and Pant, 1990). Proteoglycans from follicular fluid during

maturation of follicles were isolated and characterized (Boushehri et al., 1996). A 26.6

kDa peptide was isolated from follicular fluid of buffalo follicular fluid using 50G

Sephadex gel filtration having oocyte maturation stimulating activity (Gupta, 2002).

Sheep: Two forms of inhibin with molecular weights of 65 and 30 kDa were isolated

from follicular fluid using a combination of gel permeation chromatography, reversed-

phase high-performance liquid chromatography and preparative polyacrylamide gel

electrophoresis (Leversha et al., 1987). Gel chromatography of pooled follicular fluid

resolved two peaks of inhibitory activity (mitosis of 3T3 fibroblasts in vitro) associated

with substances of Mr approximately 180 KDa and less than 10 kDa, respectively (Carson

et al., 1988). Follicular fluid was subjected to gel-chromatography on Sephadex G-100,

and the retarded active fraction (Fr-IV, Kav = 1) was rechromatographed on G-25 to yield

3 fractions GF1, GF2 and GF3 having role in follicular maturation, ovulation and

luteinisation (Nandedkar et al., 1988). An ovarian follicular fluid peptide (OFFP) had

been identified from sheep and humans (Nandedkar et al., 1996a,b). Purification of OFFP

was achieved by ultrafiltration and gel chromatography with further purification by fast

performance liquid chromatography and reversed phase-high pressure liquid

chromatography. OFFP was a small (< 5 kDa) peptide that competed with FSH in binding

to granulosa cells in vitro and inhibited progesterone secretion from granulosa cells in

culture.

Human: A purified preparation of protein (Mr: 12-15 kDa) having inhibitory effects on aromatase activity of granulosa cells was isolated from pooled follicular fluid (Dizeraga

et al., 1983). Methanol extract of human follicular fluid was subjected to microcrystalline

thin-layer chromatography (TLC) and C18 reverse-phase high-pressure liquid

chromatography (HPLC) to yield a low molecular weight protein that stimulated human

spermatozoa motility (Fetterolf et al., 1994). A steroidogenesis inducing protein isolated

39

was found to have a potent mitogen activity for cell lines derived from ovarian surface

epithelium carcinomas (Khan et al., 1997). Zona-binding inhibitory factor-1 (ZIF-1, (Mr: 30 kDa), isolated from follicular fluid, reduced the binding of spermatozoa to the zona

pellucida (Chiu et al., 2003).

2.4. Bioassays of follicular fluid peptides Numerous attempts had been made to study the effects of follicular fluid as a whole,

as such or after charcoal treatment, follicular fluid fractions obtained by ammonium

sulphate precipitation or by membrane filtration and any purified peptide isolated from

follicular fluid by various in vivo and in vitro assays.

2.4. 1. In vivo assays Majority of the in vivo assays were conducted in laboratory animals like mice and

rats or in small ruminants like sheep and goats. Both stimulatory and inhibitory effects

were reported. Among the various in vivo assays, the follicular growth, organ growth and

differentiation in granulosa cells were studied.

2.4. 1. 1. Follicular fluid peptide FSH was reported to increase follicular growth in rats via the synthesis of a >30

kDa peptide while the growth of medium sized follicles was regulated by an oestradiol-

induced 10-kDa inhibitory peptide (Chakravorty et al., 1993). Immature mice injected

with 10 or 20 µg of ovine ovarian follicular fluid peptide (OFFP) exerted direct effect on

the granulosa cells, indicating its autocrine role in the process of follicular atresia

(Nandedkar et al., 1996b). OFFP purified from sheep enhanced apoptotic changes in

ovarian granulosa cells of mice (Rajadhyaksha and Nandedkar, 1999). Granulosa cell-

inhibitory factor (CGIF) isolated from cattle follicular fluid was found to increase the

number of small follicles but reducing the number of large follicles in rats when

administered subcutaneously at 6 g/rat (Hynes et al., 1996a). Immunization of rats and

sheep against CGIF from bovine follicular fluid increases the number of large follicles in

rats and the ovulation rate in sheep (Hynes et al., 1999). A 26.6 kDa peptide isolated from

40

buffalo follicular fluid enhanced the number of small and total number of follicles in rats

(Gupta, 2002).

2.4. 1. 2. Follicular Fluid Fractions FSH inhibitor obtained from cattle follicular fluid precipitation with ammonium

sulphate (14.5 - 18.5%) had inhibitory activity to compensatory ovarian hypertrophy in

mice (Sato et al., 1982). A porcine follicular fluid fraction suppressed the follicle

response in rats (Kling et al., 1984). Fraction obtained from sheep follicular fluid when

injected into pseudopregnant mice affect corpus luteum function, but at the end of

pseudopregnancy, the expected ovulation was suppressed. Injection of 20µg fraction into

cycling mice inhibited follicular maturation and ovulation (Nandedkar et al., 1988). The

immunosuppressive fraction isolated from cow follicular fluid inhibited mouse plaque

formation (Veselsky et al., 1991). Treatment with proteinaceous fraction of buffalo

follicular fluid delayed oestrus in goats (Agarwal et al., 1996). Fraction obtained from

buffalo follicular fluid after ammonium sulphate precipitation and column

chromatography had inhibitory activity on the compensatory ovarian hypertrophy in mice

(Srivastava and Pant, 1999). A buffalo follicular fraction (Mr: >30 kDa) caused a delay in

the onset of estrus and increased the ovulation rates in goats (Ghosh et al., 2005).

2.4. 1. 3. Whole follicular fluid Increased ovulation rate in the ewe following administration of ovine follicular

fluid was demonstrated (Cummins et al., 1983). Direct inhibition of follicular growth in

ewes by charcoal treated ovine follicular fluid was reported (Cahill et al., 1984). Adult

ewes were immunized against an inhibin-enriched preparation obtained by affinity

chromatography of bovine follicular fluid (Al-Obaidi et al., 1986). Results suggested that

the plasma of ewes immunized against inhibin-enriched preparation contained substances,

which neutralize the FSH-suppressive effects of bovine follicular fluid and inhibin-

enriched preparation in ovariectomized ewes. Heifers injected with 0.4 or 4 mg total

protein (partially purified sheep follicular fluid) was found to have a transient increase in

ovulation rate (Price et al., 1987). ). In ewes, a low dose of bovine follicular fluid (0.2 ml

subcutaneously every 8 h) had no detectable effect on the secretion of FSH, an

41

intermediate dose (0.6 ml every 8 h) depressed the plasma FSH concentration for about

24 h, and a high dose (1.8 ml every 8 h) reduced the FSH concentration to undetectable

values (Martin et al., 1987). Treatment of ewes with steroid-free follicular fluid during the follicular phase of the oestrous cycle immediately inhibited ovarian secretion of

oestradiol, inhibin, androstenedione and testosterone (Baird et al., 1990). Sheep follicular fluid contained a factor that acted directly on the ovaries to induce atresia of large

preovulatory follicles and results in immediate suppression of ovarian secretion of

inhibin, oestradiol and androstenedione (Campbell et al., 1991). Steroid-free equine follicular fluid (EFF) was used for induction of oestrus in cycling ewes and it was suggested that EFF provide a rich source of inhibin, which was biologically active in

ewes (Hernandez et al., 1997). Steroid-free ovine follicular fluid decreased ovarian steroid secretion and expression of markers of cellular differentiation in sheep (Campbell

et al., 1999b). Induction of estrus by follicular fluid in Black Bengal goats during non-

breeding season was reported (Shah et al., 1998). Total follicle population remained

unaffected but the number of non-atretic follicles decreased following buffalo follicular

fluid administration in guinea pigs (Kumar et al., 1999).

2.4. 2. In vitro assays Most of the in vitro assays were conducted using slaughterhouse ovaries of cattle,

buffalo, sheep and goats. Likewise in vivo studies, both stimulatory and inhibitory effects

were reported. Among the various in vitro assays, the oocyte development and granulosa

cell proliferation in culture were studied.

2.4. 2. 1. Follicular fluid peptide Oocyte culture system: A peptide of molecular weight 2-kDa isolated from porcine

follicular fluid was found to have an inhibitory effect on oocyte maturation (Tsafriri et al.,

1996). A high molecular weight component isolated from pig follicular fluid from

medium follicles improved male pronucleus formation rate in porcine oocytes (Naito et

al., 1990). All oocytes were arrested at the germinal vesicle stage when incubated with

42

the maturation-inhibiting protein (Mr: 60 kDa) at a concentration of 2.0 mg/ml; at a

concentration of 0.25 mg/ml, 56% of oocytes matured to the metaphase of the second

meiosis. The inhibiting fraction had no meiosis-inhibiting effect on oocytes without a

compact cumulus (Dostal et al., 1996). In a study on cumulus-enclosed cattle oocytes,

free inhibin subunit, but not inhibin A, isolated from follicular fluid, was shown to

reduce the oocyte developmental competence (Silva et al., 1999). A 26.6 kDa peptide

isolated from buffalo follicular fluid enhanced the maturation rates of oocytes both in

buffalo and sheep (Gupta, 2002, Gupta et al., 2005a).

Granulosa cell culture system: Follicular regulatory peptide isolated from porcine

follicular fluid reduced the granulosa cell aromatase activity in vitro by 50% when added

at a concentration of 25 ng/ml (Ono et al., 1986). Granulosa cells when incubated with

FSH or EGF, plus different concentrations of sheep follicular fluid peptide resulted in

specific competition of the peptide with FSH, but not with EGF, for binding to granulosa

cells (Shahid et al., 1991). Granulosa cell-inhibitory factor (GCIF), a low molecular

weight factor in cattle follicular fluid, had been shown to inhibit the proliferation of cattle

granulosa cells in vitro (Hynes et al., 1996a). Low molecular mass factors isolated from

bovine follicular fluid (Mr: 10-25 kDa) when added at the concentrations of 1, 10, 100,

1000 ng/ml in granulosa cell culture medium reduced steroidogenesis in granulosa cells in

vitro (Baratta et al., 2000). They also observed the inhibition of steroidogenesis by

purifications obtained from large, medium or small follicles (Baratta et al., 2000).

2.4. 2. 2. Follicular Fluid Fractions Oocyte culture system: The immunosuppressive fraction isolated from cow follicular

fluid reduced mitogen-induced lymphocyte proliferation in vitro. However, it had no

effect on the development of intact or zona-free oocytes and embryos (Veselsky et al.,

1991). A porcine follicular fluid fraction (Mr: 10-200 kDa) obtained by ultrafiltration, gel

filtration and ion exchange chromatography was found to enhance the maturation rates of

porcine oocytes (Yoshida et al., 1992). A porcine follicular fluid fraction (active factor <

6.5 kDa) was reported to enhance the cumulus expansion of porcine oocytes (Daen et al.,

1994). Heparin-binding fraction of bovine follicular fluid when added to the IVM

43

medium was highly effective for enhancement of developmental competence of bovine

oocytes in vitro (Ikeda et al., 1999).

Granulosa cell culture system: FSH inhibitor obtained from cattle follicular fluid

precipitation with ammonium sulphate (14.5-18.5%) suppressed FSH binding to

granulosa cells in vitro (Sato et al., 1982). Porcine granulosa cells collected from

medium-sized follicles (2-5 mm in diameter) when cultured with various concentrations

of a protein fraction (molecular weight, 12.5-16 kDa) isolated from porcine follicular

fluid, it was observed that the protein fraction at a concentration of 167 µg/ml caused a

tenfold increase in the production of progesterone, whereas a concentration of 500 µg/ml

caused a return to baseline values of progesterone in cultured granulosa cells (Battin and

DiZerega, 1985). One of seven peaks (peak 4) isolated from bovine follicular fluid was

found to inhibit the granulosa cell proliferation in vitro (Hynes et al., 1996a).

2.4. 2. 3. Whole follicular fluid Oocyte culture system: Conflicting reports were available on the effect of follicular fluid

on in vitro maturation of oocytes. A number of studies on bovine (Avery et al., 2003),

buffalo (Chauhan et al., 1997, Gupta et al., 2001a, Nandi et al., 2004), ovine (Sun et al.,

1994; Shankarappa and Reddy, 1998), caprine (Lakshmikant, 2006), porcine (Yoon et al.,

2000) and equine (Bogh et al., 2002) oocytes had shown that supplementation of the

maturation medium with follicular fluid could promote in vitro oocyte maturation and /or

in vitro fertilization and subsequent embryo development. In contrast, addition of

follicular fluid at 60% v/v (Kim et al. 1996) and 100% v/v (Ayoub and Hunter, 1993)

level inhibited oocyte maturation in cattle. Fluid from follicles 2.5-5.0 mm in diameter

showed higher meiosis inhibiting effects than that from fluid from follicles 5-10 mm in

diameter (Dostal et al., 1996). Use of cystic follicular fluid as medium for cattle oocytes

maturation was limited to a single study (Takagi et al., 1998) wherein good cumulus

expansion in cattle oocytes using cystic follicular fluid. Cystic follicular fluid though

enhancing cumulus expansion (Nandi et al., 2003b), was not capable of supporting

development of buffalo oocytes in vitro (Nandi et al., 2004).

Granulosa cell culture system: Addition of follicular fluid at 1 and 5% levels decreased

FSH-induced oestradiol production in cultured granulosa cells. FSH-induced

44

progesterone production was not affected by addition of follicular fluid (Rouillier et al.,

1998). Ovine follicular fluid was found to inhibit aromatase activity in granulosa cells.

Fractionation of follicular fluid by a desalting column demonstrated that the inhibition

was related to a compound(s) >10 kDa (Driancourt et al., 2000).

2. 4. 2. 4. In vitro follicle growth Many attempts had been made to obtain complete development of mammalian

preantral follicles in vitro. Initial studies were carried out using the mouse as a model and

resulted in the identification of various culture systems (Eppig et al., 1996a) and live pup

(Eppig and O’-Brien, 1996a). Such success could not be achieved in any domestic

animals so far. Preantral follicles were isolated by enzymatic method, mechanical method

or enzymatic pretreatment followed by mechanical isolation using fine needles (Smitz

and Cortvrindt, 2002). Follicular antrum formation in the cultured preantral follicles of

cattle (Gutierrez et al., 2000), sheep (Cecconi et al., 1999), goat (Huanmin and Yong,

2000) and pig (Wu et al., 2002) was accomplished. The time required for an antrum

formation in culture varied between species. The time required for cultured sheep

preantral follicles to grow to antral stage was 6 days (Cecconi et al., 1999, Tamilmani et

al.,2005). In contrast, no antrum was formed even after 40 days of culture in buffalo

(Gupta et al., 2002a). The growth of preantral follicles was found to be greater during 6 to

10 days of culture of buffalo preantral follicles (Nandi et al., 2005a). Buffalo antral

follicles were found to grow faster than preantral follicles in vitro (Nandi et al., 2006).

Intact preantral follicles were cultured in Minimum essential media (MEM) or

Tissue culture medium-199 (TCM-199) supplemented with serum, gonadotrophins and

growth factors (Sheep: Hemamalini et al., 2003, Tamilmani et al., 2005; Buffalo: Gupta

et al., 2002a). Endpoints in follicle culture in domestic species were intact follicle

survival, antrum formation or meiotic maturation of oocyte. The effect of follicular fluid

protein on in vitro follicle growth was not studied so far.

2. 4. 2. 5. In vitro oocyte maturation 2. 4. 2. 5. 1. Oocyte retrieval

45

Abattoir derived ovaries provide a cheap and abundant source of oocytes for in

vitro maturation. Though several methods of oocyte recovery (aspiration, slicing,

puncture of follicles) were available, slicing of the ovaries yielded maximum good quality

oocytes in sheep (Wani, 2002). Aspiration was the most widely accepted technique in

buffalo for its speed of operation (Das et al., 1996a). 2. 4. 2. 5. 2. Oocyte culture

In conventional in vitro maturation studies, oocytes were cultured in groups in 50-

100 l droplets of TCM-199 supplemented with serum and gonadotrophins at 38.5 0C in

5% CO2 for 24 hours (sheep: Wani et al.,2002, buffalo: Palta and Chauhan, 1998, Nandi

et al., 2002a). Maturation rate in vitro of oocytes are assessed by various methods like

staining the oocytes (M-II stage), identification of extruded first polar body in the

perivitelline space and degree of expansion of cumulus cell mass. Use of chemically

defined media is now recommended for oocyte culture as it negates the possible effects of

unknown components of biological fluids (Sheep: Guler et al., 2000; Buffalo: Raghu et

al., 2002a).

2. 4. 2. 5. 3. Biochemical changes in oocytes after in vitro maturation Large numbers of macromolecules like proteins are accumulated during oocyte

maturation. The mean protein content of oocytes was 0.126 µg (Grealy et al., 1996).

There was three-fold increase in protein content from immature to matured bovine

oocytes (Tomek et al., 2002). Accumulation of protein in bovine oocytes after in vitro

maturation was also demonstrated (Kastrop et al., 1990, Thompson et al., 1998). A major

change in the pattern of protein synthesis associated with resumption of meiosis was

reported (Pavlok et al., 1997). Synthesis of at least 7 oocyte specific and 5 cumulus

specific proteins were found to be stage dependant during in vitro bovine oocyte

maturation (Wu et al., 1996), Changes in the protein pattern were studied in goat oocytes

by radiolabelling of oocytes with [35S] methionine. A polypeptide of 28.1 kDa appeared

as a major band at the germinal vesicle stage and it was not found after the germinal

vesicle breakdown (GVBD) stage. Three new polypeptides (35, 36.5 and 39 kDa) were

found at GVBD and metaphase II stage (Pawshe et al., 1994a). There was an increase in

46

total protein content in buffalo oocyte after in vitro culture in media supplemented with

IGF-I (Pawshe et al., 1998, Arthi, 2003).

The information on the mineral uptake during maturation of oocytes was meager.

During in vitro maturation, calcium changed from a diffusible to a bound state, and

accumulated within the mouse oocyte (Webb et al., 2002). Calcium ions participated in

the progression of meiosis and inositol triphosphate receptor might be responsible for the

majority of Ca2+ release during bovine oocyte maturation (Li et al., 1997, Liu et al.,

1999). Gonadotrophin raised intracellular Ca2+ in sheep oocytes and the junctional

communication with the oocyte allowed a rapid diffusion of this signal into the ooplasm,

where it was involved in the resumption of meiotic division (Mattioli et al., 1998). In the

plasma membrane Ca2+ current in the immature oocyte was related to developmental

potential and calcium stores were related to morphological quality in immature oocytes

and to developmental competence in mature oocytes (Boni et al., 2002).

The phosphorus content during oocyte maturation was mostly studied in aquatic

animals (Ng et al., 1980). In buffalo, IGF-I in the oocyte culture media was shown to

stimulate the phosphorus uptake in the oocytes in the dose dependant manner (Arthi,

2003).

2. 4. 2. 6. Granulosa, cumulus and oviductal cell culture Somatic cells like granulosa cells and oviductal cells were reported to secrete

proteins and growth factors besides removing inhibitory components from in vitro cell

culture environment (Nandi et al., 2001). The metabolic cooperation between the oocytes

and cumulus cells served an important nutritive role during oocyte maturation (Tanghe et

al., 2002).

The stimulators of somatic cell growth in vitro were EGF, FGF, TGF- , PDGF,

IGFs, transferin and follicular fluids (Guraya, 1997). Granulosa cells were one of the

most extensively studied endocrinological cells for investigating the synthesis of

hormones (Vinze et al., 2004), nature of steroidogenesis (Armstrong et al., 2002) and

action of ovarian peptides (Hynes et al., 1996a). The granulosa cell monolayer was found

to support development of ovine (Rao, et al., 2002), caprine (Teotia et al., 2001), bovine

(Goodhand et al., 2000), porcine (Vatzias and Hagen 1999), bubaline (Nandi et al., 2001),

equine (Li et al., 2001) oocytes and cattle (Itoh and Hoshi, 2000) and buffalo (Ramesh,

47

2005) preantral follicles. Oviductal cell monolayer was found to increase the

developmental competence of oocytes (sheep: Holm et al., 1994, Dunford et al., 1994,

buffalo: Nandi et al., 2001, pig: Vatzias and Hagen 1999, mare: Li et al., 2001) and

embryos (sheep: Holm et al., 1994, goat: Cognie et al., 2003, cattle: Hasler et al., 2000,

buffalo: Nandi et al., 1998, pigs: Vatzias and Hagen 1999, mare: Li et al., 2001).

From the above, it is evident that the information on biochemical composition of

ovine follicular fluid was meager. Similarly, only a few studies were conducted on

bioactivities of peptides isolated from ovine follicular fluid. Information on ovine

follicular peptide(s) on folliculogenesis in vivo and in vitro, ovarian and extraovarian cell

growth is lacking. Hence the present study was undertaken with a goal to have a better

understanding of ovine ovarian physiology and increased reproductive efficiency in

sheep.

3. MATERIALS AND METHODS

3.1. Materials All chemicals used for processing of follicular fluid were obtained from Sisco

Research Lab. Pvt. Ltd., Mumbai, India, unless otherwise stated. Chemicals used for in