Development of a hamster superovulation program and adverse effects of gonadotropins on microfilament formation during oocyte development  Seung T. Lee, B.Sc., Tae M. Kim, B.Sc., D.V.M., Mi Y. Cho, B.Sc., Shin Y. Moon, M.D., Ph.D., Jae Y. Han, Ph.D., Jeong M. Lim, D.V.M., Ph.D.  Fertility and Sterility  Volume 83, Issue 4, Pages 1264-1274 (April 2005) DOI: 10.1016/j.fertnstert.2004.09.039 Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Experimental designs. Fertility and Sterility 2005 83, 1264-1274DOI: (10.1016/j.fertnstert.2004.09.039) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Morphology of embryos retrieved from golden hamsters with or without superovulation by exogenous gonadotropin. Superovulation was conducted with the administration of pregnant mare serum gonadotropin (PMSG), followed by the administration of hCG (5 or 15 IU each). Embryos, unfertilized oocytes, or pronuclear zygotes were retrieved 42 hours after natural mating. From nonsuperovulated females (A) and the females superovulated with 5 IU each of PMSG and hCG (B), only two-cell embryos were collected at retrieval. However, the major proportion were pronuclear zygotes after the treatment with 15 IU each of PMSG and hCG (C). Morphological observation showed that all were fertilized and did not have morphological abnormalities, regardless of the stage of development (×100). Fertility and Sterility 2005 83, 1264-1274DOI: (10.1016/j.fertnstert.2004.09.039) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Ultrastructure of golden hamster oocytes without (A) or with (B) treatment with pregnant mare serum gonadotropin (PMSG) and hCG. Zero (A) or 5 (B) IU each of PMSG and hCG were administered. Oocytes were examined by a transmission electron microscope at different magnifications, immediately after collection. Well-developed endoplasmic reticulum (ER) and microvilli (MV) of the ooplasmic membrane and peripherally distributed cortical granules (CG) were visible in all oocytes. The mitochondrial population in ooplasm was similar in oocytes from the 0-IU and the 5-IU treatment groups (A3, B3). Fertility and Sterility 2005 83, 1264-1274DOI: (10.1016/j.fertnstert.2004.09.039) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Confocal images of microfilament in golden hamster oocytes without (A) or with (B) treatment by 5 IU each of pregnant mare serum gonadotropin (PMSG) and hCG. Actin filament (fluorescent green color; A1, B1) was detected by palloidin staining under a confocal microscope. In pseudocolored confocal images (A2, B2), reds, oranges, and yellows represent high microfilament formation, whereas blues and greens represent relatively low formation. In oocytes from untreated hamsters, microfilament formation was comparatively evenly distributed over the whole region of the cytoplasm (A1, A2). In contrast, in oocytes of animals treated with 5 IU each of PMSG and hCG, microfilament formation was concentrated near the nucleus (N) and polar body (PB). Fertility and Sterility 2005 83, 1264-1274DOI: (10.1016/j.fertnstert.2004.09.039) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 5 Distribution and morphology of microtubule and chromosome in oocytes retrieved from golden hamsters without (A) or with (B) superovulation. Superovulation was conducted by administering 5 IU of pregnant mare serum gonadotropin (PMSG), followed by the administration of 5 IU of hCG. Monoclonal anti-α-tubulin and antifluorescein isothiocyanate conjugate clone was used for detecting microtubules (fluorescent green color), and the chromosome of oocytes (red color) was stained with propidium iodide. Confocal microscope was used for observation, and no significant changes in microtubules after superovulation treatment were detected. Fertility and Sterility 2005 83, 1264-1274DOI: (10.1016/j.fertnstert.2004.09.039) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions