Rhesus offspring produced by intracytoplasmic injection of testicular sperm and elongated spermatids Laura Hewitson, Ph.D., Crista Martinovich, B.S.,

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Rhesus offspring produced by intracytoplasmic injection of testicular sperm and elongated spermatids Laura Hewitson, Ph.D., Crista Martinovich, B.S., Calvin Simerly, Ph.D., Diana Takahashi, M.Sc., Gerald Schatten, Ph.D. Fertility and Sterility Volume 77, Issue 4, Pages 794-801 (April 2002) DOI: 10.1016/S0015-0282(01)03281-2

FIGURE 1 Selection of testicular sperm and elongated spermatids (A, B) and after injection into metaphase arrested rhesus oocytes (C–F). Testicular sperm (A) were characterized by a condensed nucleus and an enlarging flagellum with slight motility. Elongated spermatids (B) were characterized by an elongating nucleus with the migrated cytoplasm at its basal region, containing the axial filament. No visible flagellum was apparent. Pronuclear formation 15 hours after the injection of testicular sperm (C) and elongated spermatids (D) into rhesus oocytes. Cleavage stage ICSI (E) and ELSI (F) embryos used for transfer. All images were captured with a Nikon 35-mm camera attached to a Nikon TE300 inverted microscope, equipped with Hoffman modulation contrast optics. Bar = 10 μm A, B; 20 μm C, D; 30 μm E, F. Hewitson. TESA-ICSI and ELSI in non-human primates. Fertil Steril 2002. Fertility and Sterility 2002 77, 794-801DOI: (10.1016/S0015-0282(01)03281-2)

FIGURE 2 Rhesus offspring produced by the intracytoplasmic injection of testicular sperm and elongated spermatids. Tess (A) and Tickler (B) were conceived using testicular sperm and Elsie (C) was conceived using an elongated spermatid. Hewitson. TESA-ICSI and ELSI in non-human primates. Fertil Steril 2002. Fertility and Sterility 2002 77, 794-801DOI: (10.1016/S0015-0282(01)03281-2)

FIGURE 3 Microtubule and chromatin configurations of rhesus oocytes fixed 24 hours after TESA-ICSI (A, B) or ELSI (C, D). After TESA-ICSI (A) or ELSI (C), the oocyte typically remained arrested in metaphase of meiosis, characterized by an anastral, barrel-shaped spindle radially oriented at the oocyte cortex (MII, red) with chromosomes aligned across the spindle equator (MII, blue). The injected sperm (S, blue) or spermatid (S’tid, blue) did not form a male pronucleus or assemble a microtubule aster within the oocyte cytoplasm. In B, the oocyte was injected with a testicular sperm that has undergone premature chromosome decondensation resulting in both a maternal (MII) and paternal (P) meiotic spindle. The sperm tail, separated from the sperm, may still be associated with the sperm centrosome because a small aster of microtubules has formed by the tail (arrow). In D, an activated ELSI oocyte has extruded the second polar body (PB), which remains anchored to the oocyte by the mid-body (red). The decondensing female pronucleus (F) resides close to the mid-body. The spermatid is characterized by a slightly swollen nucleus associated with manchette microtubules (green, arrows) surrounding the basal region of the spermatid. Bar = 10 μm. Hewitson. TESA-ICSI and ELSI in non-human primates. Fertil Steril 2002. Fertility and Sterility 2002 77, 794-801DOI: (10.1016/S0015-0282(01)03281-2)