Summary Epidermal growth factor (EGF) has been shown to have a positive effect during oocyte in vitro maturation in several species. This study was performed to establish the capacity of equine oocytes to undergo nuclear maturation in the presence of EGF and to localise its receptor in the equine ovary by immunohistochemical methods. Oocytes were obtained by aspiration and subsequent scraping from equine follicles (15–25 mm diameter) and cultured in 3 different treatment groups for 36 h: control Group (modified TCM 199 with 0.003% BSA), EGF Group (TCM‐199 supplemented with 50 ng/ml EGF) and EMS Group (TCM 199 supplemented with 10% v/v oestrous mare serum). Each group was divided further into 3 treatments with tyrphostin A‐47, a specific tyrosine kinase inhibitor, at 0, 10 −4 and 10 −6 mmol/l. Maturation was determined as the percentage of oocytes reaching metaphase II stage at the end of the culture period. Immunohistochemical detection of EGF‐receptor (EGFR) was performed using a streptoavidinbiotin method. The recovery rate and oocyte retrieval were 84.6% (recovered oocytes/follicles aspirated) and 6.55 (oocytes/mare), respectively. Treatment with EGF significantly (P<0.05) increased the incidence of metaphase II stage compared with the control group (69.4 vs. 26.9% in controls, respectively). The specific‐tyrosine kinase inhibitor A‐47 was effective in suppressing EGF‐effect on EGF‐cultured oocytes; no significant differences were observed in EMS‐supplemented oocytes when cultured with A‐47. EGF‐receptor was localised in follicles, with localisation being more prominent in the cumulus than in mural granulosa cells. This finding, together with the increase of oocyte nuclear maturation rate when using EGF in culture media and the inhibition of maturation by tyrphostin A‐47, suggests a physiological role for EGF in the regulation of equine oocyte maturation. The results should help successful development of assisted reproductive technology in the horse.
This study evaluated the effect of a GnRH analogue conjugated to the cytotoxin, pokeweed antiviral protein (PAP), on reproductive function in adult, male dogs. Four dogs received 0.0042 mg GnRH-PAP kg(-1) hourly for 36 h, and four other dogs received 0.1 mg GnRH-PAP kg(-1) as one bolus injection daily for three consecutive days. One dog received a single bolus (0.1 mg x kg(-1)). Three adult male dogs received GnRH without the PAP conjugate, as controls. Twenty-five weeks after the initial treatment, all treated dogs received 0.1 mg GnRH-PAP kg(-1) as a single administration, whereas dogs in the control group received 0.0045 mg kg(-1) of the GnRH analogue. Serum concentrations of testosterone and LH were determined by radioimmunoassay, and testis size was measured for 9 months after treatment. Stimulation tests (5 microg GnRH kg(-1)) were used to evaluate LH release (-15, 0, 30, 60, 90, 120 min), which was assessed by measuring area under the curve. Serum testosterone concentrations were significantly lower (P<0.05) after treatment in the bolus and hourly groups than in the control group. Testosterone concentrations fell to less than 50 pg x ml(-1) in three of four dogs in the bolus group and one of four dogs in the hourly group by week 8-9 after treatment. Basal LH was lower (P<0.05) in the bolus and hourly groups than in the control group between weeks 0 and 33 after treatment. Treatment with GnRH-PAP reduced (P<0.05) LH release after GnRH stimulation in the bolus and hourly groups compared with the control group. Testis volume was lower (P<0.05) in all treated versus control dogs. In conclusion, administration of the conjugate GnRH-PAP at a 25 week interval resulted in a major disruption of reproductive parameters in male dogs; this effect was maintained for 11-12 weeks after a second injection of GnRH-PAP.
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