No abstract

Pronuclear formation, and the chromosomal constitution and developmental capacity of bovine zygotes formed by intracytoplasmic sperm injection with freeze-dried (lyophilized) spermatozoa were evaluated. Frozen-thawed spermatozoa were selected, freeze-dried, and stored at 4 degrees C until use. After 22-24 h of in vitro maturation oocytes were denuded and injected singly with a lyophilized spermatozoon. Injected oocytes were activated by treatment with 10 microM ionomycin (5 min) alone and in combination with 1.9 mM 6-dimethylaminopurine (DMAP) for 4 h. Ionomycin plus DMAP activation treatment resulted in a significantly higher proportion of sperm-injected oocytes with two pronuclei than was found after activation with ionomycin alone (74% vs. 56%; P < 0.03). The rates of cleavage, morula, and blastocyst development of sperm-injected oocytes treated with ionomycin plus DMAP were higher than after activation with ionomycin alone (63.3%, 34.2%, and 29.6% vs. 44.7%, 18.7%, and 10.6%, respectively; P < 0.05). Seventy-three percent of blastocysts produced with lyophilized sperm were diploid. These results demonstrate that in vitro-matured bovine oocytes can be fertilized with freeze-dried sperm cells, and that resultant zygotes can develop into karyotypically normal blastocysts.

The overall aim of this investigation was to examine the expression and steroidal regulation of insulin-like growth factor-I (IGF-I) and the IGF-I receptor in the rat corpus luteum and to examine the specificity of IGF-I action in the two luteal cell populations. We first examined whether the corpus luteum expresses the IGF-I and IGF-I receptor genes. Using a solution hybridization/RNase protection assay, IGF-I and IGF-I receptor mRNAs were represented by protected bands 224 and 265 bases in length, respectively. In addition, Northern blot analysis showed that, as in liver, rat IGF-I and IGF-I receptor cDNAs hybridized with 7.5-, 1.8-, and 0.8- to 1.2-kilobase transcripts and an 11-kilobase transcript, respectively. Both IGF-I and IGF-I receptor mRNAs were detected on all days of pregnancy tested (days 5-21). Since the rat corpus luteum increases remarkably in size and steroidogenic capacity at midpregnancy due to estradiol stimulation, we determined whether these developmental changes are accompanied by an increased expression of the IGF-I and/or IGF-I receptor genes. Total RNA was isolated from corpora lutea of day 12 hypophysectomized-hysterectomized rats treated with or without estradiol for 3 days. Estradiol caused a clear and marked reduction in IGF-I and IGF-I receptor mRNA. [125I]IGF-I bound with high specificity and affinity to luteal cell membranes. Large and small cell populations forming corpora lutea of day 3 and 14 pregnant rats were separated by elutriation and used for the determination of binding activity and for cell culture, respectively. IGF-I receptors were found to be localized principally in the large luteal cell population. The small luteal cells had approximately 6.5-fold less IGF-I-binding activity. The difference in binding activity in both cell populations was reflected in the ability of both cell types to respond to IGF-I. IGF-I (25 ng/ml) had a profound effect on the production of progesterone by the large luteal cells. No stimulatory effect of IGF-I on the small luteal cells was observed. Addition of estradiol (10 ng/ml) to the cell culture remarkably enhanced IGF-I stimulation of progesterone biosynthesis by the large luteal cells. In summary, the results of this investigation have revealed that the corpus luteum of the pregnant rat is a major site of expression of both the IGF-I and IGF-I receptor genes.(ABSTRACT TRUNCATED AT 400 WORDS)