Incorporation of a GnRH agonist, leuprolide acetate, into regimens with exogenous gonadotropins to produce ovarian stimulation and ovulation in the nonpregnant squirrel monkey.

This study was designed to measure the effects of variations in the length of pretreatment with a GnRH agonist, leuprolide acetate (LA), on subsequent follicular development and ovulation. The hypothesis was that the duration of LA suppression of pituitary function does not adversely affect ovarian response to standardized ovulation induction protocols in squirrel monkeys. The first phase determined the dose and duration of LA needed to achieve a hypogonadal state. One of two groups received daily subcutaneous injections of 50 microg of LA. The other received a single injection of 175 microg of a depot suspension of LA. Sera were assayed for estradiol (E2) and progesterone (P). E2 and P levels increased 2- to 5-fold with peak levels on days 4 and 7, respectively. Suppression of steroid levels took 10 to 15 days in the LA-treated group. Depot-LA did not effectively suppress steroid production. After suppression, females receiving daily LA received five daily injections of hMG to stimulate follicular development. E2 and P increased in these animals. These results suggest that cycling squirrel monkeys have P-secreting capacity throughout the cycle. This may explain how the squirrel monkey is able to accommodate both a short (4-5 day) luteal phase of their 9 day cycle and implantation from 5 to 7 days after ovulation. A second study compared exogenous follicle stimulating hormone (FSH) to endogenous gonadotropins released as a response to LA in ovulation induction. Steroid production and hCG-induced ovulation were assessed. LA treatment was compared to a standard ovulation induction protocol by using a randomized cross-over measures design. There were no differences in E2 and P levels in response to dosages of either LA or hMG. The ovulatory response following LA treatment was not significantly greater than that using FSH. The number of animals with unovulated, large follicles was greater on the FSH protocol (12/18) compared to the LA protocol (3/18). Thus, a single injection of a depot preparation of LA is sufficient to stimulate follicular development and ovulation when followed by an hCG injection. Based on this observation and the data on unovulated large follicles, it is suggested that the ovary responds more readily to endogenous gonadotropins released by LA than to exogenous FSH.

Cryopreservation of murine zygotes for use in testing culture environments.

Developing one-cell mouse zygotes are more sensitive to in vitro environmental conditions than are cleavage-stage embryos. However, for convenience and reproducibility, cryopreserved two-cell zygotes are routinely used for such assays. Concern over the possibility of inducing damage by exposing one-cell zygotes to cryoprotective agents and freeze-thaw procedures during syngamy led us to examine one-cell zygotes, with and without visible pronuclei, in an effort to minimize or avoid these effects and obtain the highest possible developmental rate. In vivo fertilized mouse zygotes were collected 21 to 43 h after administration of human chorionic gonadotropin (hCG). Suspensions of zygotes in 2M ethylene glycol were aspirated into 0.25-ml plastic insemination straws and slowly cooled at -0.5 degree C/min to -40 degrees C before being plunged into liquid nitrogen for storage. Zygotes were thawed, rinsed, and placed in culture. Zygotes were examined initially for damage from the freeze-thaw procedure. Daily in vitro development was recorded. In this group of zygotes, no damage was apparent immediately after thawing, and a high degree of development in vitro was observed. Thus, usefulness of a cryopreservation method for one-cell murine zygotes has been confirmed.

Effect of sperm dose on pregnancy rate from intrauterine insemination: a retrospective analysis.

Both the number of motile spermatozoa inseminated and the site of insemination have been correlated with the probability of pregnancy in patients inseminated with donor sperm cells from fertile men. Nevertheless, more data on the minimum sperm dose required to achieve a pregnancy are needed to understand this apparent relationship. We analyzed retrospectively 2280 cycles of intrauterine insemination to test the hypothesis that intrauterine insemination requires a minimum number of motile sperm cells to maximize the pregnancy rate. Our analysis of 1761 cycles of intrauterine insemination using from 200,000 to more than 200 million motile sperm cells showed no significant relationship between sperm dose and pregnancy rate.

Absence of a direct effect of recombinant tumor necrosis factor-alpha on human sperm function and murine preimplantation development.

OBJECTIVE: This study was designed to test the in vitro effects of human recombinant tumor necrosis factor (rTNF) on sperm motility, fertilization, and preimplantation development. DESIGN: A sensitive enzyme immunoassay was used to determine half-lives of rTNF and confirm concentrations of cytokine throughout experimental conditions. Effect of rTNF on human sperm survival was measured by computer-assisted methodology, and effect on human sperm penetration was assessed by hamster ova penetration. Cytokine effect on murine gamete interaction was determined by in vitro fertilization (IVF). Murine preimplantation development was assessed by in vitro development of cryopreserved-thawed one-cell zygotes. RESULTS: The half-life of rTNF was reduced by the addition of sperm to culture media (P less than 0.001). Sperm motility (P = 0.245) and hamster ova penetration (P = 0.62) were not altered by incubations in the presence of concentrations of rTNF up to 10,000 U/mL. Mouse IVF (P = 0.60) and preimplantation development (P = 0.56) were not altered by rTNF in concentrations up to 5,000 U/mL. CONCLUSIONS: These results demonstrate rTNF by itself does not interfere with gamete function or early embryo development.

Fertilization: a uterine glycosaminoglycan stimulates the conversion of sperm proacrosin to acrosin.

Acrosin is a proteinase required for mammalian fertilization, and in freshly ejaculated spermatozoa exists as an inactive zymogen, proacrosin. A factor prsent in uterine flushings of gilts stimulates the conversion of highly pruified boar proacrosin to acrosin. Characterization of this factor indicates that its active component is a glycosaminoglycan.

Effects of prostaglandins and catecholamines on rat spleen adenylate cyclase in vitro.

The results reported here show some characteristics of adenylate cylase (EC 4.6.1.1) derived from homogenates of rat spleen, and describe the in vitro stimulation of this enzyme by prostaglandins, nucleotides, and F- under conditions where cyclic nucleotide degradative pathways are effectively inhibited. Particulate fractions from rat spleen homogenates contain high adenylate cyclase activities, and the highest specific activity is recovered in a particulate fraction prepared by low speed (1200 X g) centrifugation. Activity found in all particulate fractions is stimulated by fluoride, prostaglandins E1 and E2, catecholamines, and purine nucleotides. No stimulation is caused by prostaglandins F1 alpha and F2 alpha. Stimulation by prostaglandin E1 and E2 is augmented by GTP and other purine nucleotides, and stimulation by the combination of GTP and prostaglandin E1 is equal to that caused by optimal fluoride concentrations. Stimulation caused by L-isoproterenol is additive to that caused by GTP but is not increased by GTP.

Stimulation of adenylate cyclase from isolated hepatocytes and Kupffer cells.

Hepatocytes and Kupffer cells were separated from rat liver after prelabeling the Kupffer cells with colloidal iron and perfusion of the liver with digestive enzymes. The activity of several enzymes from Kupffer cells and hepatocytes was compared to validate this method of cell separation. The ratios of hepatocyte to Kupffer cell specific activities of glucose-6-phosphatase, 5'-nucleotidase, adenylate cyclase, and acid phosphatase were 20, 0.39, 0.18, and 0.078, respectively. Adenylate cyclases from hepatocytes and Kupffer cells were stimulated by fluoride ion, GTP, and catecholamines. Hepatocyte adenylate cyclase was also stimulated by glucagon, secretin, vasoactive intestinal polypeptide, and by prostaglandin E1, whereas, the Kupffer cell enzyme was completely insensitive to these hormones. The stimulation of hepatocyte adenylate cyclase by combinations of glucagon plus secretin, or glucagon plus vasoactive intestinal polypeptide, were equivalent to the sum of the individual stimulations. This suggests that the hepatocyte has specific receptors for glucagon and for vasoactive intestinal polypeptide and secretin. Prostaglandin E1 stimulation of hepatocyte adenylate cyclase was not additive to the stimulation caused by polypeptide hormones or catecholamines, nor did prostaglandin E1 decrease stimulation caused by these hormones. Although prostaglandin-sensitive adenylate cyclase was recovered with hepatocytes, 40 to 50% of the total liver prostaglandin-sensitive activity was recovered in a fraction of cell debris mixed with small cells which did not phagocytize colloidal iron.