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C. M. Lubicz-Nawrocki and M. C. Chang

Summary. Spermatozoa in the vas deferens of the hamster lose their fertilizing capacity 3 days after ligation of the initial part of the duct and after 2 days if the testes are removed at the time of ligation. Sham-ligation had no effect on the fertilizing life of vasal spermatozoa on the contralateral side even 3 days after bilateral castration. Unilateral castration for 3 days had no effect on the fertilizing capacity of spermatozoa from the ipsilateral unobstructed duct, whereas no eggs were fertilized by spermatozoa from the contralateral ligated duct associated with the remaining testis. Unlike testosterone, 5α-dihydrotestosterone injected daily or implanted subcutaneously in Silastic tubes maintained normal fertilizing capacity for 2 days in castrates and for 3 days in intact males. Within each group, ligation had no effect on the level of fructose in the seminal vesicle on that side compared with the level in the gland on the other side.

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N. S. Martus, H. G. Verhage, P. A. Mavrogianis and J. K. Thibodeaux

The purpose of this study was to determine the effect of a partially purified bovine oviductal glycoprotein (bOGP) on fertilization rates of bovine oocytes. The effect of albumin (control protein) or bOGP at 100 μg ml−1 during the 16–18 h fertilization period was evaluated in a standard IVF system using a sperm concentration between 0.5 and 0.125 × 106 spermatozoa ml−1. bOGP maintained a higher (P < 0.05) fertilization rate (62.0% versus 31.2%) at 0.125 ×106 spermatozoa ml−1 compared with the albumin control. The enhancement of fertilization by bOGP was blocked by the inclusion of a specific antibody to bOGP, whereas the antibody with albumin had no effect. A 2 h gamete preincubation step was subsequently included in the IVF procedure (0.125 × 106 spermatozoa ml−1) to determine whether the effect of bOGP was mediated through an interaction with the oocyte, the spermatozoon or both. When oocytes were preincubated with bOGP the fertilization rates were higher (P <0.05) than with the albumin control (oocytes and spermatozoa exposed to albumin), whereas preincubation of spermatozoa with bOGP did not affect fertilization rates. There was no synergistic effect of preincubating oocytes and spermatozoa with bOGP. The increase in fertilization rate achieved by preincubating oocytes with bOGP was blocked with a specific antibody to bOGP. These results suggest that the increase in fertilization rates observed when bOGP is included during the 16–18 h fertilization period are primarily mediated through the interaction of bOGP with the oocyte since the same facilitatory effect was observed with a 2 h preincubation of oocytes before IVF. The ability to block these effects with a polyclonal antibody specifically generated against bOGP shows that this biological activity is due to bOGP. In summary, bOGP enhances fertilization in bovine oocytes whether it is included during preincubation or insemination and this appears to be due to a direct effect on the oocyte.

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Cytological examination of the oocytes of a number of mammalian species has indicated that they will resume meiosis upon liberation from the Graafian follicle into a simple culture medium (Chang, 1955; Edwards, 1962, 1965a, b). Evaluation of the physiological status of such oocytes after periods of maturation would best be tested by noting their ability to undergo fertilization, cleavage and, ultimately, to produce viable young. Recent experiments have attempted in-vitro fertilization of mammalian eggs matured in culture and, whilst this has apparently yielded some success with human material (Edwards, Bavister & Steptoe, 1969) and in the rabbit (Thibault & Gérard, 1970), the contrary has proved the case in the large domestic species. In many experiments involving cow and pig eggs, sperm penetration of the zona pellucida of artificially matured oocytes was never

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Monika Fluks, Katarzyna Szczepanska, Takao Ishikawa and Anna Ajduk

In fully grown ovarian follicles both transcriptionally active (NSN) and inactive (SN) oocytes are present. NSN oocytes have been shown to display lower developmental potential. It is possible that oocytes that have not completed transcription before meiosis resumption accumulate less RNA and proteins required for their further development, including those responsible for regulation of Ca2+ homeostasis. Oscillations of the cytoplasmic concentration of free Ca2+ ions ([Ca2+]i) are triggered in oocytes by a fertilizing spermatozoon and are crucial for inducing and regulating further embryonic development. We showed that NSN-derived oocytes express less inositol 1,4,5-triphosphate receptor type 1 (IP3R1), store less Ca2+ ions and generate weaker spontaneous [Ca2+]i oscillations during maturation than SN oocytes. Consequently, NSN oocytes display aberrant [Ca2+]i oscillations at fertilization. We speculate that this defective regulation of Ca2+ homeostasis might be one of the factors responsible for the lower developmental potential of NSN oocytes.

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J. Motlík and J. Fulka

Summary. Rabbit ovarian oocytes co-cultured with granulosa cells were transferred for fertilization to the oviducts of recipient does. Oocytes with cumulus cells and membrana granulosa cells were isolated before or 3 h after hCG injection. Granulosa cells did not prevent the resumption of meiosis but the time sequence of nuclear maturation was retarded by about 3 h. When oocytes were isolated from FSH-stimulated ovaries and cultured with only their cumulus cells or with cumulus and granulosa cells of the same origin, non-decondensed sperm heads were detected in about 30% oocytes after fertilization. Culture of FSH-stimulated oocytes with granulosa cells isolated 3 h after hCG injection substantially enhanced the development of male pronuclei to 88·2% and regular cleavage to 85·2% 10 h and 20 h after transfer, respectively. Further improvement was observed if the oocytes and their co-cultured granulosa cells had been exposed to hCG for 3 h.

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A Talmor-Cohen, R Tomashov-Matar, E Eliyahu, R Shapiro and R Shalgi

The earliest visible indications for the transition to embryos in mammalian eggs, known as egg activation, are cortical granules exocytosis (CGE) and resumption of meiosis (RM); these events are triggered by the fertilizing spermatozoon through a series of Ca2+ transients. The pathways, within the egg, leading to the intracellular Ca2+ release and to the downstream cellular events, are currently under intensive investigation. The involvement of Src family kinases (SFKs) in Ca2+ release at fertilization is well supported in marine invertebrate eggs but not in mammalian eggs. In a previous study we have shown the expression and localization of Fyn, the first SFK member demonstrated in the mammalian egg. The purpose of the current study was to identify other common SFKs and resolve their function during activation of mammalian eggs. All three kinases examined: Fyn, c-Src and c-Yes are distributed throughout the egg cytoplasm. However, Fyn and c-Yes tend to concentrate at the egg cortex, though only Fyn is localized to the spindle as well. The different localizations of the various SFKs imply the possibility of their different functions within the egg. To examine whether SFKs participate in the signal transduction pathways during egg activation, we employed selective inhibitors of the SFKs activity ((PP2 and SU6656). The results demonstrate that RM, which is triggered by Ca2+ elevation, is an SFK-dependent process, while CGE, triggered by either Ca2+ elevation or protein kinase C (PKC), is not. The possible involvement of SFKs in the signal transduction pathways that lead from the sperm–egg fusion site downstream of the Ca2+ release remains unclear.

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A. M. Miller, M. E. Roelke, K. L. Goodrowe, J. G. Howard and D. E. Wildt

Summary. Eight female pumas were treated i.m. with 1000 (N = 5) or 2000 (N = 3) i.u. PMSG followed 84 h later by 800 i.u. hCG. Eggs were recovered 24–26 h after hCG from ovarian follicles by using laparoscopy and transabdominal aspiration. Mature eggs were inseminated in vitro 4–6 h later whereas immature eggs were cultured for 24 h and then inseminated. Electroejaculates from 3 pumas were diluted with mKRB before insemination to evaluate the influence of sperm concentration on fertilization. Seven of 8 pumas responded with follicle development, and 140 eggs were recovered from 145 follicles (96·6%; 77 mature, 43 immature, 20 degenerate eggs; mean ± s.e.m., 20·0 ± 5·9 eggs/female). Overall fertilization rate was 43·5% (total eggs fertilized = 40) despite using inseminates containing 82·99% pleiomorphic spermatozoa. Of the 36 immature oocytes matured in vitro and inseminated, 12 were fertilized even though 50% of the inseminating spermatozoa contained an acrosomal defect. Fertilization rate of mature oocytes collected from follicles appeared unrelated (P > 0·05) to PMSG dose or number of spermatozoa/inseminate. This study demonstrates that a high proportion of follicular eggs can be recovered laparoscopically from adult pumas treated with PMSG and hCG. These gametes are capable of being fertilized in vitro (immediately or after maturation in vitro) even with low quality semen with a high incidence of sperm pleiomorphisms.

Keywords: in vitro fertilization; puma; oocyte maturation; teratospermia

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P. Jimena, J. A. Castilla, F. Peran, R. Molina, J. P. Ramirez, M. Acebal, F. Vergara and A. Herruzo

Summary. This study was undertaken to evaluate the relationship between concentrations of insulin and insulin-like growth factor I (IGF-I) in follicular fluid and fertilization and cleavage of human oocytes fertilized in vitro. The concentration of oestradiol, progesterone, luteinizing hormone, follicle-stimulating hormone, testosterone, insulin and IGF-I was determined in 36 follicular fluids, free of visible blood contamination and containing mature oocyte–corona–cumulus complexes, obtained from 12 women undergoing in vitro fertilization. Follicular development was induced by clomiphene citrate and human menopausal gonadotrophin, and follicular aspiration was performed 35 h after an ovulatory dose of human chorionic gonadotrophin. Concentrations of IGF-I were significantly higher in follicular fluids associated with mature oocytes that fertilized and cleaved, than in follicular fluid associated with mature oocytes that did not fertilize (P < 0·001). There was no difference in the concentration of insulin between follicular fluids from which fertilized oocytes were obtained and those with oocytes that remained unfertilized. No significant correlations were found between rates of embryo cleavage, concentrations of insulin and IGF-I. Multiple linear regression analysis demonstrated that the concentrations of IGF-I in follicular fluid were predicted statistically by a negative regression coefficient for the concentration of testosterone, and by a positive regression coefficient for the concentration of progesterone in follicular fluid. No candidate variable was included in the model to predict concentrations of insulin. These data suggest an important role for IGF-I in the mature follicle.

Keywords: follicular fluid; insulin; insulin-like growth factor I; in vitro fertilization; human

Free access

Y. Fukui, A. M. Glew, F. Gandolfi and R. M. Moor

Summary. The present experiments were designed to identify possible male-specific effects on early embryonic development in vitro. Sheep oocytes were matured in vitro for 24–26 h and then fertilized in vitro using equal numbers of viable spermatozoa from 1 of 6 Clun Forest rams. At 15–18 h after insemination, oocytes were either fixed and examined for fertilization and polyspermy or further cultured in modified M 199 medium for 3 days in an oviduct epithelial co-culture system. There were significant differences in 5 separate trials between the rams with respect to the rate of fertilization, degree of polyspermy and cleavage rate after monospermic fertilization. The mean rate of fertilization varied from 89% in Ram B to 43% in Ram C while the percentage of polyspermic eggs varied from 5 to 34%. Both the absolute number of embryos cleaving to the 16-cell stage and the calculated percentage of monospermic eggs reaching the 16-cell stage differed markedly between groups of eggs fertilized by different rams. The results indicate that the development of sheep eggs in vitro is differentially affected by the ram from which the spermatozoa are collected.

Keywords: ram effect; in-vitro fertilization; embryo development

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Fernanda González Echeverría, Patricia S. Cuasnicú, Alejandra Piazza, Lucrecia Piñeiro and J. A. Blaquier

Summary. The fertility of spermatozoa from the different epididymal segments of hamsters was tested by in-vivo insemination. Caput and proximal corpus spermatozoa were non-fertile; spermatozoa from the distal corpus epididymidis fertilized 13% (38/290) oocytes and those from the proximal and distal cauda epididymidis 71 and 87%, respectively. When tested by in-vitro insemination, distal corpus spermatozoa penetrated 44% of oocytes while those from the distal cauda fertilized 87% of oocytes. Spermatozoa from the distal corpus recovered in Medium BMOC fertilized 13% (28/219) of oocytes in vivo, while those mixed with an epididymal protein preparation (0·8 mg protein/ml) fertilized 24% (49/204; P < 0·01) of oocytes.

When distal corpus spermatozoa were inseminated in vivo with 0·8 mg epididymal protein preparation 34% (31/90) oocytes were fertilized and only 22% (23/103; P < 0·05) oocytes were fertilized when the proteins were obtained from epididymides of animals castrated for 30 days. When distal corpus spermatozoa were preincubated for 5 h in medium without (control) or with protein preparation (0·8 or 1·6 mg protein/ml), a significant increase in in-vitro oocyte penetration was found (25 compared with 45%; P < 0·05) when the protein was present at 1·6 mg/ml.

These results confirm and extend previous observations suggesting a role for androgen-dependent glycoproteins secreted by the epididymis in the acquisition of fertilizing ability that occurs during sperm maturation.