Dynamics of Follicular Fluid in One-humped Camel (Camelus dromedarius)
Contents
In the present study, ovarian follicular fluid and serum biochemical, hormonal, electrolytes and amino acids profiles in female dromedary camel (Camelus dromedarius), were investigated. Fluid from small (2–6 mm) and large follicles (7–20 mm) and blood samples were collected from 25 clinically healthy adult female camels. The concentrations of glucose, cholesterol, triglycerides, high-density lipoproteins, urea, total proteins, albumin, globulin, fibrinogen, alanine aminotransferase, aspartate aminotransferase and tri-iodothyronine were lower (p ≤ 0.05) in large follicles when compared with the small follicles. However, the concentrations of low-density lipoproteins, uric acid, creatinine, alkaline phosphatase and acid phosphatase in small and large follicles did not differ. The concentrations of oestradiol 17-β and progesterone were higher (p ≤ 0.05) in large follicles. The serum concentrations of these hormones were many folds lower (p ≤ 0.05) than those of follicular fluid. Among electrolytes, the concentration of phosphorus was higher (p ≤ 0.05) in the large follicles, while that of potassium and chloride were lower (p ≤ 0.05) in the small follicles. Serum concentrations of sodium, chloride, calcium and phosphorous were higher (p ≤ 0.05), while that of potassium lower (p ≤ 0.05) than corresponding concentrations in the follicular fluid. The concentrations of leucine and arginine were higher (p ≤ 0.05) in follicular fluid when compared with serum concentrations, while the reverse was true for other amino acids. In conclusion, this study is indicative of either low or high concentrations of certain biochemical metabolites, hormones, electrolytes and amino acids in small and large follicles for the individual roles that they play in the growth and development of follicles in the one-humped she-camel.
Introduction
The camel is gaining popularity in many countries of the world because of its ability to survive and perform well under arid and semi-arid climatic conditions. At present, there are approximately 27 096 millions heads of camels in the world (Al-Ani 2004). Most of these camels are in African, Middle-Eastern and South-East Asian countries. A camel can produce approximately 1700–3600 l of milk in a lactation period of 270–540 days, and 125–480 kg of meat per animal (Al-Ani 2004). Camels also provide hides and bones, which are a good source of foreign exchange earning for a country. Furthermore, among different species of livestock, the camel remains the most neglected species in the field of scientific research.
Within the ovarian follicle, the developing oocyte is surrounded by the follicular fluid, which is a serum transudate modified by follicular metabolic activities. Besides meeting nutritional requirements of the developing oocyte, follicular fluid also maintains a proper environment for the maturation of the oocyte. It is an avascular compartment within the mammalian ovary and separated from the perifollicular stroma by the follicular wall that constitutes a blood–follicle barrier (Bagavandoss et al. 1983).
Besides a serum transudate, follicular fluid also contains locally produced substances that share the metabolic activity of follicular cells (Gerard et al. 2002). This metabolic activity, together with the barrier properties of the blood–follicle barrier, has been shown to change significantly during the growth phase of the follicle (Bagavandoss et al. 1983; Gosden et al. 1988). Therefore, biochemical composition of follicular fluid can differ in small and large follicles. Variations in follicular fluid concentrations of metabolites can also affect the quality of the oocytes within the follicles themselves.
Before focusing on possible effects of metabolic changes within the follicle on oocyte quality, it seems necessary to investigate physiological concentrations of some common metabolites in their fluid from small and large follicles, and determine to what extent the serum and follicular fluid concentrations of these metabolites are correlated. Therefore, this study was carried out to investigate ovarian follicular fluid from small (2–6 mm) and large (7–20 mm) follicles and serum biochemical, hormonal, electrolytes and amino acids profiles in the female dromedary camel (Camelus dromedarius).
Materials and Methods
Sample collection
Ovaries were recovered from 25 adult she-camels (C. dromedarius), 7- to 11-year olds, with clinically normal reproductive organs after slaughtering. The camel is known to be an induced ovulator (Skidmore et al. 1995) and the corpus luteum was seen only in pregnancy or after an infertile mating (El-Wishy 1992). As non-pregnant animals were included in the present study, very few of them had corpora lutea on their ovaries. The ovaries were placed in plastic bags and transported to the laboratory on ice. Within an hour after slaughtering, the diameters of the ovarian follicles were measured for each female with the help of the vernier calipers. Based on the diameters, the follicles were arbitrarily categorized as small (2–6 mm) or large (7–20 mm). Follicles more than 20 mm in diameter were considered as cystic (Tibary and Anouassi 1997) and were not included in the current study. Similarly, haemorrhagic and atretic follicles were identified macroscopically following the criteria of Kruip and Dielman (1982) and were not sampled. Fluid from each healthy follicle was aspirated by means of sterilized 22-guage hypodermic needles and syringes. The follicular fluid collected from the small follicles was pooled from the same ovary of the same animal. Yet, for estimation of amino acids, the fluid of small and large follicles was pooled. The follicular fluid was centrifuged at 1252 × g at 4°C for 10 min, after which the supernatant was harvested and stored in small aliquots at −20°C for further analysis.
Before slaughtering, jugular blood was also collected from each she-camel using sterilized 18-gauge hypodermic needles and syringes. The blood was centrifuged at 1252 × g at 4°C for 10 min. The harvested serum was stored in small aliquots at −20°C for further analysis. No pre-slaughter information about the nutritional and reproductive status of the camels was available.
Sample analysis
Follicular fluid and serum samples were analysed in triplicate for various biochemical metabolites. Glucose, cholesterol, triglycerides, high-density lipoproteins (HDLs), low-density lipoproteins (LDLs), uric acid, creatinine, urea, total proteins, albumin, globulin, fibrinogen, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and acid phosphatase (ACP) were measured using commercially available kits. Hormones including oestradiol 17-β, progesterone, tri-iodothyronine (T3) and thyroxin (T4) were measured using commercially available ELISA kits. The sensitivities of assay for oestradiol 17-β, progesterone, T3 and T4 were 16 pg/ml, 0.05 ng/ml, 0.2 ng/ml and 0.4 μg/ml, respectively. The average intra-assay and interassay coefficients of variation were kept under 10% for all investigated hormones. The cross-reactivity of the oestradiol 17-β assay was 0.05% for estriol and 0.2% for estrone, while it was negligible for corticosterone, progesterone and dehydroepiandrosterone. The samples were spiked with the known concentration of oestradiol-17-β in a ratio of 1 : 1 and percentage recovery was ranged from 86% to 97%. The cross-reactivity of progesterone assay was 0.10% for oestradiol 17-β and testosterone, and 0.02% for cortisol. Tri-iodothyronine and T4 concentrations were measured after adding a known concentration of T3 and T4 standards into the samples and recovery percentage, calculated, was 86–101% and 88–102%, respectively. Cross-reactivity was found to be zero for 3,3′,5 tri-iodothyroacetic acid and 3,3′,5 tri-iodothyropropionic acid. The T3 and T4 are made up of single amino acids, thus the ELISA antibodies were also specific to camel T3 and T4.
The concentrations of sodium and potassium were measured using a flame photometer (Jenway PFP7 Industrial Flame Photometer, Garforth, Leeds Ls25 IDX, UK), while calcium and phosphorus were measured using an atomic absorption spectrophotometer (SpectrAA 5; Varian Techtron, Mulgrave, Victoria 3170, Australia). The chloride was measured by titration of samples against silver nitrate solution (Sawyer et al. 1994). Amino acids analysis was performed by automatic amino acid analyser (Moore and Stein 1954).
Statistical analysis
Mean values (±SE) for the concentrations of various biochemical constituents in the fluid from small and large follicles and blood serum were computed. To see the magnitude of variation between two follicular groups and blood serum, the data were subjected to analysis of variance using a completely randomized design (Steel et al. 1997). Duncan’s multiple range test (Duncan 1955) was applied for multiple mean comparisons, where necessary, using the minitab Computer Package. Amino acid contents of follicular fluid and serum were compared using the t-test.
Results
Biochemical profiles
Glucose concentration was higher (p ≤ 0.05) in fluid from small follicles when compared with the serum, while it was significantly lower (p ≤ 0.05) in fluid from large when compared with the small follicles. Low-density lipoprotein cholesterol concentration was significantly (p ≤ 0.05) higher in serum when compared with the fluid from follicles of either diameters. The fluid from small follicles had significantly higher (p ≤ 0.05) concentrations of triglycerides, total proteins, albumin, globulin, fibrinogen, ALT and AST when compared with the fluid from large follicles and serum, except the concentrations of fibrinogen and AST in the serum where they were significantly lower (p ≤ 0.05). The concentration of serum cholesterol, HDL cholesterol and urea were significantly higher (p ≤ 0.05) when compared with the fluid from small follicles, and the concentrations of these parameters tended to be significantly lower (p ≤ 0.05) in fluid from large follicles when compared with the fluid from small follicles. The concentrations of creatinine, uric acid, alkaline and ACPs did not differ among the serum or fluid from small and large follicles (Table 1).
| Parameters | Small follicles (2–6 mm) | Large follicles (7–20 mm) | Serum |
|---|---|---|---|
| Glucose (mg/dl) | 142.50 ± 4.96a | 39.14 ± 6.74c | 90.14 ± 7.55b |
| Cholesterol (mg/dl) | 3.60 ± 0.83b | 1.62 ± 0.40c | 128.4 ± 5.5a |
| Triglyceride (mm/l) | 0.75 ± 0.23a | 0.51 ± 0.02b | 0.54 ± 0.08b |
| HDL (mm/l) | 0.48 ± 0.20b | 0.37 ± 0.08c | 0.81 ± 0.15a |
| LDL (mm/l) | 0.24 ± 0.06b | 0.25 ± 0.05b | 0.35 ± 0.09a |
| Uric acid (mg/dl) | 2.30 ± 0.18 | 1.60 ± 0.11 | 2.75 ± 0.15 |
| Creatinine (mg/dl) | 1.05 ± 0.05 | 1.22 ± 0.37 | 2.38 ± 0.49 |
| Urea (mg/dl) | 43.88 ± 6.80b | 29.00 ± 3.42c | 63.50 ± 5.73a |
| Total proteins (g/dl) | 8.49 ± 0.84a | 6.21 ± 0.62b | 6.76 ± 0.29b |
| Albumin (g/dl) | 4.87 ± 0.30a | 3.51 ± 0.28b | 3.59 ± 0.11b |
| Globulin (g/dl) | 3.81 ± 0.76a | 2.70 ± 0.69b | 3.17 ± 0.28ab |
| Fibrinogen (mg/dl) | 1.09 ± 0.22a | 0.71 ± 0.09b | 0.37 ± 0.08c |
| ALT (IU/l) | 45.9 ± 3.6a | 28.7 ± 1.44b | 35.8 ± 3.4ab |
| AST (IU/l) | 138.3 ± 2.46a | 106.9 ± 1.80b | 77.60 ± 3.10c |
| Alkaline phosphatase (IU/l) | 98.00 ± 12.59 | 92.60 ± 3.58 | 114.50 ± 8.40 |
| Acid phosphatase (IU/l) | 3.39 ± 0.33 | 4.04 ± 0.65 | 6.40 ± 0.36 |
- Values are expressed as mean ± SE (n = 25).
- a–cDenote significant difference (p ≤ 0.05) in rows.
- HDL, high-density lipoprotein; LDL, low-density lipoprotein; ALT, alanine aminotransferase; AST, aspartate aminotransferase.
Hormones
The concentrations of hormones including oestradiol 17-β, progesterone and T4 were lower (p ≤ 0.05) in the serum when compared with the fluid from either follicular class, while oestradiol 17-β and progesterone was significantly (p ≤ 0.05) higher in fluid from the larger follicles instead of the smaller follicles. Between the follicle classes, the fluid from small follicles had significantly higher (p ≤ 0.05) T3 and T4 concentrations when compared with the fluid from large follicles (Table 2).
| Hormones | Small follicles (2–6 mm) | Large follicles (7–20 mm) | Serum |
|---|---|---|---|
| Oestradiol 17-β (pg/ml) | 56.8 ± 1.2b | 138.0 ± 8.1a | 20.41 ± 1.8c |
| Progesterone (ng/ml) | 121.9 ± 4.2b | 141.1 ± 4.3a | 0.98 ± 0.48c |
| Tri-iodothyronine (ng/ml) | 5.46 ± 0.71a | 3.35 ± 0.58b | 1.30 ± 0.29c |
| Thyroxin (μg/ml) | 189.6 ± 5.6a | 147.2 ± 3.97a | 84.3 ± 4.89b |
- Values are expressed as mean ± SE (n = 25).
- a–cDenote significant difference (p ≤ 0.05) in rows.
Electrolytes
The concentrations of serum sodium, chloride, calcium and phosphorus were significantly higher (p ≤ 0.05) when compared with the fluid from follicles of either diameters. Perhaps, the concentration of chloride was significantly lower (p ≤ 0.05) in the fluid from large follicles when compared with the fluid from small follicles and the concentration of phosphorus was significantly lower (p ≤ 0.05) in fluid from the smaller ones when compared with the larger. The potassium concentration was significantly higher (p ≤ 0.05) in fluid from small follicles, while it was significantly lower (p ≤ 0.05) in serum (Table 3).
| Electrolytes | Small follicles (2–6 mm) | Large follicles (7–20 mm) | Serum |
|---|---|---|---|
| Sodium (mEq/l) | 137.05 ± 3.6b | 148.7 ± 2.5ab | 178.24 ± 2.92a |
| Potassium (mEq/l) | 13.53 ± 0.71a | 8.11 ± 0.43b | 4.90 ± 0.25c |
| Chloride (mg/l) | 121.60 ± 6.60b | 102.70 ± 9.89c | 173.47 ± 4.29a |
| Calcium (mg/dl) | 7.52 ± 0.47b | 9.42 ± 2.61b | 13.30 ± 0.86a |
| Phosphorus (mg/dl) | 3.97 ± 0.68c | 6.49 ± 1.40b | 12.47 ± 1.29a |
- Values are expressed as mean ± SE (n = 25).
- a–cDenote significant difference (p ≤ 0.05) in rows.
Amino acids profiles
Follicular fluid concentrations of all the amino acids measured in the current study in camels, except leucine and arginine, were lower (p ≤ 0.05) than the serum. Concentrations of glutamic acid and cysteine were relatively higher than other amino acids except leucine in follicular fluid (Table 4).
| Amino acids (mg/dl) | Follicular fluid | Serum |
| EAA | ||
| Threonine | 0.0057 ± 0.0006b | 0.5069 ± 0.0950a |
| Valine | 0.0016 ± 0.0004b | 0.0671 ± 0.0093a |
| Methionine | 0.0035 ± 0.0002b | 0.2113 ± 0.0020a |
| Isoleucine | 0.0078 ± 0.0009b | 0.3789 ± 0.0512a |
| Leucine | 0.0335 ± 0.0039a | 0.0169 ± 0.0033b |
| Phenylalanine | 0.0086 ± 0.0009b | 0.5778 ± 0.0078a |
| Lysine | 0.0026 ± 0.0004b | 0.1168 ± 0.0027a |
| Histidine | 0.0010 ± 0.0001b | 0.6003 ± 0.0332a |
| NEAA | ||
| Asparagine | 0.0079 ± 0.0003b | 0.7853 ± 0.0053a |
| Serine | 0.0055 ± 0.0006b | 0.2820 ± 0.0375a |
| Glutamic acid | 0.0117 ± 0.0014b | 0.1450 ± 0.0042a |
| Alanine | 0.0025 ± 0.0002b | 0.0309 ± 0.0038a |
| Cysteine | 0.0125 ± 0.0013b | 0.0411 ± 0.0057a |
| Tryptophan | 0.0074 ± 0.0008b | 0.0355 ± 0.0014a |
| Arginine | 0.0048 ± 0.0003a | 0.0021 ± 0.0002b |
| Glycine | 0.0078 ± 0.0002b | 0.4790 ± 0.0067a |
- Values are expressed as mean ± SE (n = 25).
- a,bDenote significant difference (p ≤ 0.05) in rows.
- EAA, essential amino acids; NEAA, non-essential amino acids.
Discussion
Biochemical profiles
Glucose plays an important role in ovarian metabolism because it is the major energy source for the ovary. Perhaps this is because the small follicles have the ability to filter and reserve the high concentrations of glucose from blood for utilization in their development to the mature Graafian follicle. A decrease in a concentration of glucose with the development of follicles also implies that the consumption of glucose increases in large follicles. Glucose and metabolic hormones have been shown to act directly at the ovarian level to regulate the steroidogenesis (Williams et al. 2001; Munõz-Gutiérrez et al. 2004). Gerard et al. (2002) and Iwata et al. (2004) observed a decrease in the glucose concentration in the fluid of pre-ovulatory follicles developing from a dominant follicle in mares and cows, respectively. However, in dairy cows Leroy et al. (2004) observed an increase in the glucose concentration of follicular fluid with an increase in the follicle size. In addition, they also reported that glucose concentration was significantly lower in follicular fluid when compared with the serum.
Cholesterol is known to be a precursor of all steroid hormones, including oestrogen and progesterone in females (Hafez 1993). The low cholesterol concentration in the fluid from large follicles indicated biotransformation of cholesterol to sex steroids, i.e. oestrogen and progesterone, and in the present study the concentrations of these sex steroids were higher (p ≤ 0.05) in larger follicles when compared with the smaller ones (Table 2). It is interesting to note that the concentration of cholesterol in the serum was many folds higher (p ≤ 0.05) than in the fluids from follicles of either size. It also suggests that serum cholesterol might not be the major metabolic compound available for steroidogenesis in ovarian follicles. Endresen et al. (1990) reported that granulosa cells have a large store of cholesteryl esters that may provide free cholesterol for the pre-ovulatory progesterone or steroidogenesis.
Triglycerides are the storage form of lipids, and their hydrolysis yields one molecule of glycerol and three molecules of fatty acids, and the energy needed for the growing follicle. Thus, continuous and rapid utilization of triglycerides might have resulted in their low concentrations in large compared with small follicles. A similar trend was found in the triglycerides concentration, with a higher triglycerides concentration in the fluid from smaller than the larger follicles in porcine and bovine ovaries, as reported by Chang et al. (1976) and Leroy et al. (2004).
High-density lipoproteins are transudated from blood into the follicles and provide cholesterol to the granulosa cells for steroidogenesis (Le Goff 1994). The low HDLs concentrations in large follicles indicate excessive turnover of HDLs to cholesterol and ultimately to the sex steroids. In healthy growing follicles, HDLs maintain progesterone production at a low rate (Volpe et al. 1991). Thus, HDLs might support steroidogenesis during follicular growth and prevent cholesterol accumulation in pre-ovulatory follicles. The HDL protein Apo A1 may also be involved in the activation of Lacithin Cholesterol Acyltransferase (LCAT) enzyme (Acton et al. 1996), which is responsible for cholesterol reverse transport (Johnson et al. 1991) and the synthesis of the most potent steroidal oestrogens in the follicular fluid (Pahuja et al. 1995).
The blood–follicle barrier permits the passage of increasing amounts of high molecular weight proteins such as LDLs only during the pre-ovulatory period (Rom et al. 1987). Thus, the LDL molecules enter the antrum of the pre-ovulatory follicles only during the final phase of the follicle development (Enk et al. 1986) and deliver a large amount of cholesterol for progesterone synthesis. The lower LDL concentrations in both the follicle classes might be due to the impermeability of the blood–follicle barrier to this compound.
In addition, low LDL concentration in both the follicles with respect to serum and HDLs in the follicular fluid might be a part of the defence mechanism of the follicle. High concentrations of LDLs are responsible for more progesterone synthesis (Porta et al. 1999). The latter reduces the oocyte’s fertilization potential (Volpe et al. 1991) or induces its atresia (McNatty et al. 1985) by inhibiting oestradiol 17-β production (Erickson 1995).
The results of uric acid and creatinine concentrations in the current study are in accordance with the results reported by Chang et al. (1976) in porcine ovarian follicles. The presence of uric acid in the follicular fluid indicates that it might be responsible for an antioxidant activity, because uric acid has been reported to be an important water-soluble antioxidant (Knapen et al. 1999).
The higher urea concentration, the end product of protein metabolism, in the current study in small follicles indicates that protein catabolism is higher in small than large follicles. Leroy et al. (2004) also observed a decrease in urea concentration when the follicles grew from small to large size. The ALT and AST catalyse reversible biotransformation reactions between alanine and glutamic acid, and aspartic acid and glutamic acid, respectively. The higher activity of ALT and AST in small follicles recorded in this study (described in a later section) would lead to more production of glutamic acid and ultimately produce more urea, as glutamic acid is converted into urea with aspartic acid and ammonia as intermediate products.
In swine, total proteins concentration in the fluid from small follicles was slightly higher than in fluid from large follicles, while the serum had non-significantly higher total proteins concentration compared with small and large follicles (McGaughey 1975). Furthermore, serum contained less albumin and α-globulins compared with the follicular fluid, while gamma-globulins were higher in serum than in the follicular fluid. The higher albumin concentration in the fluid from the smaller follicles rather than the larger follicles in the present study is in agreement with the results reported for follicular fluids from pigs (McGaughey 1975). Albumin also plays a vital role in the development of a colloidal pressure which might contribute to the high viscosity of the follicular fluid. It is a fact that the quantity of follicular fluid increases with follicular growth and before ovulation, which is accompanied by a moderate increase in osmolarity in the ovulatory follicle (Smith and Ketteringham 1938). It can be speculated that albumin may increase osmotic pressure and the movement of solvent, i.e. water, and contribute towards the low concentration of albumin in the fluid from large follicles. According to McGaughey (1975), fast migrating α-globulins were higher in small follicles and appeared to be correlated with the meiotic division of the oocytes. The high globulin concentration in the fluid from small follicles observed in the current study in the camels might be the result of more fast migrating α-globulins when compared with the fluid from large follicles. David et al. (1973) reported that in rabbits, the protein concentrations of follicular fluid and serum did not differ significantly. The sieving function of the blood–follicle barrier that prevents the diffusion of the plasma proteins can be speculated to become more effective with the growth of the follicles. Increased levels of high molecular weight proteins in follicular fluid were reported to be associated with poor oocyte quality, fertilization failure or follicular atresia in the buffalo (Talevi et al. 1994).
Fibrinogen is a high molecular weight protein (Mr: 400 000) and the low fibrinogen in large follicles could either be the result of the dilution effect in large follicles, blockage of its diffusion into the follicular fluid during later stages of follicular growth, or increased consumption by the larger follicles. Strickland and Beers (1976) have proposed that plasminogen activators are secreted into the follicular fluid where they convert the plasminogen into plasmin, which is involved in tissue remodelling during ovulation. Fibrinogen is one of the proteins that act as a mediator of plasminogen activators (Neiuwenhuizen et al. 1983).
In cattle oocytes, the metabolism of glutamine has been shown to increase with oocyte maturation (Rieger and Loskutoff 1994). Thus, in the present study, the high concentrations of ALT in small follicles indicate the higher requirement of glutamine during the early stages of growth. The findings of AST distribution in current study are in agreement with those reported by Chang et al. (1976) in porcine. The AST catalyses reversible biotransformation reactions between aspartic and glutamic acid. Therefore, high concentration of this enzyme in the fluid from small follicles further supports the view that glutamic acid is much more essential during the early stages of follicular growth.
The ALP and ACP play a vital role in follicular growth as well as atresia. Phosphatases are constituents of follicular fluid from cows (Henderson et al. 1990) and pigs (McGaughey 1975). Alkaline phosphatase was observed to be involved in active transport in bovine follicular tissue (Kenney 1973). Dimino and Elfont (1980) suggested that ACP might be important in steroidogenesis.
Hormones
The results of the current study imply that the concentrations of oestradiol 17-β and progesterone increased with the growth of the follicles. Low serum concentrations of these hormones suggest that follicular cells are the main site for synthesis and release of these sex steroids. In cattle, LH stimulates the theca interna cells to secrete testosterone, which is then converted into oestrogen by the granulosa cells under the influence of FSH (Hafez 1993). In camel, follicular growth produces a peak of oestradiol 17-β in the serum (Skidmore et al. 1996). Oestradiol 17-β, alone, has little effect on granulosa cells of maturing follicles, but it was required for maximum FSH stimulation of Cyp19 expression and oestradiol synthesis (Adashi and Hsueh 1982; Zhuang et al. 1982; Fitzpatrick and Richards 1991; Tetsuka and Hillier 1996), LH receptor expression and LH responsiveness (Kessel et al. 1985; Knecht et al. 1985; Segaloff et al. 1990), antrum formation (Hirshfield 1991; Wang and Greenwald 1993), gap-junction formation (Burghardt and Anderson 1981) and prevention of atresia (Billig et al. 1993). The optimum folliculogenesis activity of oestrogen was dependent upon oestrogen receptor-β functions, because these receptors appeared to facilitate follicle maturation from the early antral to the pre-ovulatory follicular stages (Emmen et al. 2005).
The direct actions of progesterone have been known to enhance the response of cultured rat granulosa cells to FSH by increasing cAMP (Goff et al. 1979) and inhibiting apoptosis in large granulosa cells in culture (Peluso and Pappalardo 1994). Zalanyi (2001) also suggested that progesterone was involved in stimulating ovulation in the human. Progesterone has also been shown to enhance the production of proteolytic enzymes important for the rupture of follicles at ovulation (Iwamasa et al. 1992) either directly or by enhancing endometrial relaxin production (Yki-Jarvinen et al. 1985) which was thought to stimulate the release of proteases by granulosa cells (Too et al. 1984). Yet, higher progesterone concentrations in large follicles suggested that this hormone would be readily available for its biotransformation into testosterone and, hence, into oestrogen.
Thyroid hormones are essential for normal reproductive functioning of the ovaries and follicular growth, as hypothyroidism has been shown to be associated with impaired fertility in women (Longcope 1991). In a monolayer culture system of porcine granulosa cells, Maruo et al. (1987) demonstrated that thyroid hormones could be synergized with FSH to exert direct stimulatory effects on granulosa cell functions, including LH receptor formation and induction of steroidogenic enzymes, such as 3β-hydroxysteroid dehydrogenase and aromatase. The receptors of thyroid hormones in the granulosa cells have been reported in pigs (Wakim et al. 1987) and rats (Bandyopadhyay et al. 1996).
Electrolytes
Iwata et al. (2004) reported that sodium concentration was insignificantly different between fluids from small and large follicles in bovine, while serum sodium concentration was found to be higher than small follicular fluid in pigs (Chang et al. 1976). Iwata et al. (2004) and Leroy et al. (2004) also observed higher potassium in smaller follicles than larger in bovine. In swine, higher potassium in small compared with large follicles was attributed to glucose utilization, a process that leads to the transfer of potassium from extracellular fluid to intracellular sites (Chang et al. 1976). Perhaps the same is true for the camel, an idea that is supported by the fact that higher glucose utilization was observed in large follicles in this study.
In dairy cows, Leroy et al. (2004) observed a decrease in chloride concentrations with the advancement in follicular growth. The chloride was known to initiate the LH-stimulated steroidogenesis in the chicken granulosa cells (Morley et al. 1991), human chorionic gonadotropin-stimulated steroidogenesis in oocytes of amphibians (Skoblina and Huhtaniemi 1997) and steroidogenesis in adrenal glands by influencing the cAMP production (Cooke et al. 1999). Yet, the chloride ions were responsible for enhancing the activity of angiotensin-converting enzyme, a metalloenzyme, which had been discovered in the follicular fluid of the porcine ovaries (Matsui and Takahashi 2002).
Iwata et al. (2004) reported that calcium concentration did not differ between the small and large follicles in bovine. The higher serum calcium in follicular fluid may be related to the fact that serum is a major pool for many minerals that are utilized by various tissues. Calcium is required for normal functioning of the granulosa cells (Leung and Steele 1992) and steroidogenesis in granulosa cells during in vitro studies (Eckstein et al. 1986). Phosphorus is known to be a vital part of cAMP, as the second messenger in physiological action of steroid hormones (Hafez 1993). The concentration of cAMP increases with the maturation of follicles in pigs (Chang et al. 1976). Thus, higher phosphorus concentration in large follicles can be related to a high demand of cAMP.
Amino acids profiles
Amino acids, like other compounds such as glucose, cholesterol and electrolytes, are prerequisites for the maturation and fertilization of bovine oocytes in vitro. The oxidative metabolism of amino acids is the major energy-generating pathway in bovine oocytes (Rieger and Loskutoff 1994). The lower concentrations of most amino acids in follicular fluid may reflect the utilization of both essential and non-essential amino acids by the cells located between the dense capillary network of the follicle and the follicle’s interior. Alternatively, it may reflect transport characteristics across these cells. It is likely that both factors contribute to creating this concentration gradient.
Jozwik et al. (2006) reported that the concentrations of branched-chain amino acids, including isoleucine, leucine and valine, increased in the fluid from pre-ovulatory ovarian follicles in humans. In the placenta, the utilization of branched-chain amino acids is a major source of ammonia production (Jozwik et al. 1999). It is thought that the amino group produced from transamination reaction involving branched-chain amino acids may be used for glutamate synthesis (Jozwik et al. 1999). In the rat model, intraperitoneal injection of 14C-leucine resulted in uptake and prolonged radioactivity of this amino acid isotopes in follicular fluid (Yatvin and Leathem 1964). The arginine is a non-essential amino acid and is readily consumed by the proliferating cell (Sakagami et al. 1998). Its higher concentration in the follicular fluid, rather than in the serum, can be attributed to its excessive demand by the proliferating cells of the ovarian follicles. Glutamine is involved in a variety of intracellular metabolic pathways. In pre-implantation mouse embryos, uptake and utilization of glutamine have been reported (Chatot et al. 1990). Thus, the relatively high availability of glutamic acid in the follicular fluid can be speculated for its bioconversion into glutamine. Cysteine, the reduced and active form of cystine, is required for the formation of coenzyme A, proteins with cross-linked polypeptide chains, metallotionine and enzymes with active sulfhydryl (SH–) groups (e.g. glutathione peroxidase, Na/K-ATPase). Relatively high concentration of cysteine can be speculated to be involved in these biochemical processes in the follicles.
In conclusion, the present study is indicative of either low or high concentration of certain biochemical metabolites, hormones, electrolytes and amino acids between small and large follicles and the individual roles that they play in the growth and development of follicles in the one-humped she-camel.
