TDK conference 2014

Introduction: Gonadal development starts in the early embrionic stage. Functional testis is formed 14 days after conception, differentiation from an indifferentiated gonad to testis is completed at 35th days in dogs which is then followed by their descent. There are limited studies about testicular steroid hormone production of dog foetus in advanced pregnancy. The aim of our study was to evaluate steroid hormone levels in the serum of the mother and the foetuses, foetal fluids (amnion, allantois) and in foetal gonadal tissues.

Materials and method: Testosteron (T), dihydrotestosteron (DHT), progesterone (P4), 17β-oestradiol (E2) concentrations were measured from six healthy pregnant bitches. Hormone concentrations including T, DHT, E2 were measured by ELISA kits (DRG Instruments GmbH, Marburg, Germany) and P4 was measured by QuantiCheck (SZIE, Budapest, Hungary). Hormone levels of foetal testicles and ovaries were determined by preparing tissue homogenates (TH). Homogenates were created by a tissue homogenizator (Janke & Kummel Ika-Werk UltraA-Turrax). Tissues were homogenizated with adequate proportions of PBS solution. Homogenates were centrifuged for 15 minutes with 5000 rpm. Supernatants were collected and stored at -20 °C until measurements.

Results. Samples obtained from six pregnant bitches resulted in n=41 samples. Foetal gender distribution was 19 females and 22 males. Steroid hormone concentration of gonadal TH were magnitudes higher than in foetal serum and foetal fluids (P4: 12-1208 ng/ml, T: 27.3-4784 ng/ml, DHT: 20,79-2834065,8 pg/ml, E2: 132,5-2596 pg/ml). Grouping on the basis of gender, we found that foetal serum DHT concentrations were higher in males (1042,26-2834065,8 pg/ml) than in females (20,79-79578 pg/ml) and T concentrations in THs as well (female: 27.3-106.5 ng/ml and male: 800-1600 ng/ml). Samples from the bitch with 40-45 days long pregnancy yielding one female and five female foetuses showed different Testosteron levels hormone levels exclusively in TH (female: 27,3-163,2 ng/ml, male: 570,4-4784 ng/ml). Mostly higher steroid concentrations were determined in maternal serum concentration in the dam with one female foetus than that with more foetuses. Concentrations of P4 and E2 did not differ in any sample.

Conclusions. Our results suggest intensive hormone production in foetal gonads. Results also refer to high activity of steroidogenic enzymes in the background. Exclusively high DHT level from foetal serum and T level from THs may allow differentiation of male and female foetuses. We measured extremely high concentrations of T and DHT in the THs of both genders. The function of high intragonadal steroid concentrations is unclear but regulatory role during the development and maturation is presumable. Further examinations are needed to clarify the paracrine and endocrine effect of steroid production by gonads during intrauterine development.