Progesterone Regulation of Primordial Follicle Assembly in Bovine Fetal Ovaries

Eric E. Nilsson1* and Michael K. Skinner1

1Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman WA 99164-4231ABSTRACT

Fertility in mammals is dependant on females having an adequate primordial follicle pool to supply oocytes for fertilization. The formation of primordial follicles is a process known as ovarian follicular assembly. In rats and mice Sex steroids have been shown to inhibit follicle assembly both in vitro and in vivo, with assembly occurring after birth when the pups are removed from the high-steroid maternal environment. In contrast, primordial follicle assembly in other species, such as cattle and humans, occurs during fetal development before birth. The objective of the current study is to determine if progesterone levels regulate primordial follicle assembly in fetal bovine ovaries. Ovaries and blood were collected from bovine fetuses and correlated to the developmental age of the fetus. The mid-gestation stages between 62 and 170 days were used. Primordial follicle assembly directly correlated with this stage of fetal development ranging from 5% (62d) to 85% (170d) assembled follicles. Interestingly, ovarian progesterone concentration was found to decrease with increasing fetal age and correlated to increased primordial follicle assembly. Ovarian estrogen levels also decreased and correlated with fetal age. Fetal bovine ovary cultures were used to investigate the direct actions of progesterone. Treatment with progesterone significantly decreased primordial follicle assembly. Observations indicate that sex steroids regulate ovarian primordial follicle assembly in cattle, as they do in rodents. The speculation is made that the progesterone inhibition of primordial follicle assembly is critical to prevent premature development of follicles during gestation and this is conserved from rodents to humans.

Introduction•Oocytes are stored in mammalian ovaries in an arrested state in structures called primordial follicles.

•During the formation of primordial follicles (follicle assembly) groups or ‘nests’ of oocytes separate to form individual primordial follicles.

•Follicle assembly is inhibited by maternal progesterone or estrogen in rodents, but in cattle and humans assembly occurs before birth while maternal sex steroids are present.

Question: Do sex steroids regulate primordial follicle assembly in fetal calf ovaries?

Experimental approach:

•Collect fetal bovine ovaries from slaughterhouses.

•Characterize the timing of follicle assembly in fetal bovine ovaries.

•Measure fetal ovary progesterone and estrogen concentrations, and fetal blood progesterone.

•Culture fetal ovary fragments with and without progesterone.

Conclusions• Fetal ovarian progesterone and estrogen concentrations decreased

during mid-gestation, at the same time as follicle assembly was initiated.

• This occurred at a time during which maternal blood progesterone and estrogen levels were stable.

• In cultured fetal bovine ovaries, treatment with progesterone significantly decreased follicle assembly.

• These data indicate that progesterone, and possibly estrogen, are important regulators of ovarian follicle assembly.

Figure 1: Fetal bovine ovarian follicle assembly and development is shown at different gestational ages, indicated left margin. Representative fetal ovaries were collected at different gestational ages, sectioned and hematoxylin/eosin stained. The left column of images were taken at 100x microscope magnification, and the right column taken at 400x magnification from the same section. Arrows = oocyte nests. Arrowheads = individual assembled follicles

Figure 2: The percentage of oocytes in an ovarian cross-section that are assembled into primordial follicles at different gestational ages (days). Regression line is from a sigmoidal dose-response equation with variable slope (R2=0.91).

Figure 6: Correlation of progesterone concentration in fetal bovine ovaries and the percentage (%) of oocytes assembled into primordial follicles as a function of gestational age (days). Follicle assembly is initiated as ovarian progesterone concentration declines.

Figure 3: The progesterone (A) and estrogen (B) concentration in fetal bovine ovaries (ng/g wet weight) taken from fetuses at different gestational ages (days). Regression lines are fitted using exponential decay equations. R2=0.77 for progesterone regression; R2 =0.73 for estrogen regression.

Figure 5: Representative ovarian cortex piece after ovary culture showing oocyte nests and assembled follicles. Image on right is a duplicate of left image with the addition of green shaded areas showing oocyte nests, and arrows pointing to assembled primordial follicles. Microscope magnification is 400x.

Figure 4: The effect of progesterone on follicle assembly in cultured fetal bovine ovaries. Fetal ovary cortex pieces were cultured in the absence (control) or presence of progesterone (10-6M). After culture the pieces were sectioned, stained and the proportion of assembled follicles determined. The percentage of assembled follicles was normalized to the control culture percentage of assembled follicles for each experimental replicate, for each fetal ovary (n=4). * = p<0.05 that treatment is different than control by one-sample t-test.