Changes in Number of Granulosa Cells , Follicular Fluid Levels and Diameter of Oocytes during Folliculogenesis in Pre-pubertal Gilts at Marketing Weight

The follicles (1.8 to 7.8 mm in diameter) were recovered from the ovaries in marketed pigs and the number of granulosa cells, the diameter of oocytes obtained from different development stages of the follicles and follicular fluid levels were determined. Correlations between size measurements and cell counts as well as the diameter of antral follicles and oocytes were also investigated. The results indicated that, while expanding in size, follicle numbers decreased with a greater atretic proportion. Granulosa cells increased in numbers continuously and remained unchanged beyond the size of 200 mm in non-atretic follicles, whereas a sudden drop of granulosa counts was observed in atretic follicles. Follicular fluid, on the other hand, linearly increased its volume with follicle size and differed little between those of non-atretic and atretic follicles. Diameters of oocytes in non-atretic follicles increased to its maximum when follicles expanded to 150 mm and maintained its size during later follicular expansion. It is concluded that, for in vitro culture, the optimal size of porcine follicle should be between 150 to 180 mm if they are collected from pre-pubertal gilts of marketing size slaughtered in an abattoir. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 12 : 1647-1651)


INTRODUCTION
The oocytes and follicles of a porcine embryo begin to form on day 18 and the primordial germ cells are observed in genital ridges (Black and Erickson, 1968).Normally, the primordial follicles are surrounded by a single layer of cells (Oxender et al., 1979) and the primary follicle is a potential ovum surrounded by a single layer of cuboidal granulosa cells with a diameter of around 0.5 mm (Chiquoine, 1960;Guraya, 1985).In rabbits, cumulus-oocyte complexes (COCs) collceted from ovarian follicles can be cultured and parthenogenetically activated in vitro if its diameter is above 1 mm, although maturation remained low (Ju et al., 2002).On prenatal pigs, the secondary follicle is an ovum surrounded by two or more layers of granulosa cells with a diameter of 101.7±41.8µm and, if continue to develop, oocyte will expand its size from 45.6 to 120 µm in diameter (Black and Ericson, 1968;Motlik et al., 1984;Hyttel et al., 1997).Usually, a follicle with a diameter of up to 3 mm containing an oocyte with a diameter from 80 to 110 µm is referred to as the tertiary follicle, or is called a Graffian follicle with a fluid-filled antrum and potential ovum in gilts (Christenson et al., 1985;Hyttel et al., 1997).The granulosa cells of Graffian follicles are rich in FSH receptors and regulated by FSH to secret estrogens (Leung and Armstrong, 1980;Huang et al., 2002).Under the influence of FSH and estrogens, the Graffian follicles soon develop to pre-ovulate follicles with a diameter of 7 mm or more (Loeken and Channing, 1985), and contain oocytes with a diameter above 120 µm.The follicles are in a consistent state of physiological and histological changes during the maturation stages.
In porcine genetic engineering, information regarding the follicular fluid, number of granulosa cells, and diameter of oocytes is essential, but it has not been well established yet.In this present study, atretic and non-atretic follicles are collected from marketing gilts in abattoir to reveal basic data of the follicular fluid, number of granulosa cells, and diameter of oocytes.Those information is of value for in vitro manipulation of germ cells.

Recovery of antral follicles
Ovaries were collected from carcass offal immediately removed from slaughtered pigs in a local abattoir and immersed into saline and carried to a laboratory at the room temperature.After arrival, single follicles were isolated from ovaries by two pliers to unrip connective tissue between follicles by mechanical force.Transparent or opaque follicles, as described by Grimes and Ireland (1986), were classified into atresia and non-atresia categories and their greatest diameter were measured by a micro ruler.into tubes to centrifuge at 600×g for 15 min.After follicular fluid volume were measured, sediments were separated and were suspended into phosphate buffer solution, so that COCs can be separated by micro pipette, and their diameter with membrane, zona pellucida and cumulus oophorus can be measured under microscope with a micro ruler.

Recovery and count of granulosa cells
Selected follicles of different size were punched and granulosa cells were collected and suspended in 1 ml of saline with methalene blue for 10 min.An aliquot (10 µl) was placed on a heamocytometer to count granulosa cells under a reverse microscope.

Statistical analysis
Data were presented by mean and standard deviation.Significant differences between means was tested by analysis of one-way variance (ANOVA) and their, correlation between variables was computed by using statistical software of SAS, 1986.

Number of follicles
The follicles observed ranged from 1.9 to 7.8 mm in diameter, average weight of the collected ovaries was 2.3±0.8g and the number of non-atretic and atretic follicles in the ovaries were showing Figure 1.It can be seen that the total number of non-atretic follicles decreased with increase in size; on the other hand, the atretic follicle was remained in low number, but slightly increased in the last stage of size increase and rendered the atretic to non-atretic ratio increased along with increasing size.Only 8% of can be classified as small follicles with volume of 60 mm 3 atretic as volume of follicle was 180 mm 3 , an almost equal number of atretic and non-atretic follicles was found.Follicular size of 180 mm 3 in pre-puberty gilts seems is a critical size in follicle growth at which decreased of granulosa cells and oocyte diameter occurred.This corresponded with the description by Wang and Greenwald (1991), for the ratio of healthy and atresia follicles obtained from the ovary mouse and suggesting a common mechanism may exists in mammals.In cyclic cows, the atretic follicle is always higher then non-atretic follicles.This result did not correspond with our present study, indicating once reached puberty a different mechanism controls follicular growth.

Number of granulosa cells
The correlation of the number of granulosa cells in atretic and non-atretic follicles are shown in Figure 2. The number of granulosa cells in atretic follicles was correlated with size of follicular size in a best fitting of y= -0.8X 2 +248X+7,778 (r=0.99); while it was y=-0.5X 2 +212X +9,870 (r=0.99) for non-atretic follicles.Two regressions illustrated a different pattern; the number of granulosa cells of the atretic follicle increased along with follicle volume reached 150 mm 3 , maintained as follicle volume expanded to 180 mm 3 , and drastically decreased thereafter.In the nonatretic follicles, granulosa cell number also increased accompanies follicle volume expansion up to 220 mm 3 and maintained even reached 250 mm 3 (Figure 2).The drop of cell numbers in large atretic follicles suggests a substantial apoptosis, which did not see in non-atretic follicles.In Figure 2, it is also observed that before follicle volume developed to 150 mm 3 , number of granulosa cells differs little between atretic and non-atretic follicles, the later drop in number of granulosa cells in atretic follicles beyond 150 mm 3 suggests that a part of the granulosa cell is already in a process of apoptosis in atretic follicles during development and resulted a dramatically decrease in cell numbers once it reached to a certain stage of development.

Follicular fluid
The follicular fluid amounts of atretic and non-atretic antral follicles in different follicle size are shown in Figure 3.The increase in follicular fluid level is linearly accompanied by an increase in follicle volume in either atretic or non-atretic antral follicles and no significant difference between them.Thus, follicular fluid levels, despite atretic or non-atretic, increase in associated with the expansion of follicle diameter.However, a small but not significant gradual higher fluid level was observed from size of 115-120 mm 3 onward in the atretic follicles suggesting a compensation of fluid fill in atretic when cells started to undergo apoptosis.

Oocyte diameter
The diameter of oocytes in atretic and non-atretic in the antral follicles was shown in Figure 4 and 5.The average diameter of oocytes lacking pellucida, in both atretic and non-atretic, of the antral follicles was development to 175-190 mm 3 of the follicle volume.As Matas et al. (1996) described, the diameter of an oocyte in pigs can be divided into four groups: less than 105 µm, between 105 to 115 µm, between 116 to 120 µm, and above 120 µm.After being cultured in vitro, the penetration ability of the sperm in the larger groups was higher.However, in bovine, the diameter of the follicles is not related to the rate of sperm penetration and fertility either the male pronucle forming ability (Leibfried-Rutledge et al., 1985).These data suggest that once meiosis is initiated, the development ability in vitro differed little at least in bovine oocytes.On the other hand, the cleavage rates (more than 3 divisions) of the nuclear transplant rabbit embryos were greater in the small nuclear donors than in the large donors.It appeared that no difference in the development competence between large and small isolated blastomeres was observed (Ju et al., 2003).
In gilts, the average diameter of oocytes containing zona pellucida in atretic and non-atretic antral follicles is similar (Figures 4 and 5) and both were maximum when developed up 20-160 mm 3 of the follicle volume.These data clearly indicated that oocytes increase its size with the expansion in follicle diameter.Also, in those atretic oocytes became slightly larger than in non-atretic presumably some in the atretic follicle were in their degeneration process showing less incompleteness physicality.This result observed from porcine was similar to those described by Arlotto et al. (1996) and Fair et al. (1995) in bovines that the expansion in diameter of the oocytes was accompanied by an increase in follicle diameter and when antral follicle diameter reached beyond 4 mm there was no more further changes in diameters of the oocytes.Suzuki et al. (1994), on the other hand, reported that the diameter of oocytes from immature bovines was larger than those from matured ones after insemination.The causes were not clear.The diameter of the oocytes increases rapidly before the follicle forms a small antral and the rapid synthesis of protein or DNA by the oocytes took place while the diameter was less than 110 µm.After that, the synthesis of protein or DNA nearly stopped (Fair et al., 1995(Fair et al., , 1996)).In gilts, Christmann et al. ) (y=-0.0038x 2 +1.2336x+152.49,R 2 =0.6039); content zona pellucida ( ) (y=-0.0019x 2 +0.5544x +124.65,R 2 =0.377) and non-zona pellucida ( ) (y=-0.0015x 2 +0.5256x+90.237,R 2 =0.4766).) (y=-0.0063x 2 +1.4,984x+150.37,R 2 =0.6614); content zona pellucida ( ) (y=-0.0014x 2 +0.4559x +129.41,R 2 =0.4492) and non-zona pellucida ( ) (y=-0.0013x 2 +0.4652x+94.98,R 2 =0.4653).