Expression of GnRH receptor in canine corpus luteum, and luteal function following deslorelin-induced puberty delay D Kaya*, A Gram*, S Aslan, S Schäfer-Somi, M Kuru, MP Kowalewski   (*) contributed equally

Background GnRH modulates both inhibitory and stimulatory responses affecting ovarian function in dogs and other mammals The inhibitory actions of GnRH or its agonists (GnRH-a) on gonadal steroidogenesis involve suppression of gonadotropin receptors or mediator enzymes involved in steroidogenic pathways. Is the main regulator of pituaitary gonadotrophs. Deslorelin effects contraception by temporarily suppressing the reproductive endocrine system, preventing production of pituitary (FSH and LH) and gonadal hormones (estradiol and progesterone in females and testosterone in males). Implanted deslorelin appears active in suppressing the function of the pituitary-gonadal axis and thus sexual activity for 8-12 months, although longer periods of anoestrus have been reported.

Background In dogs, the steroidogenic activity of CL seems to be controlled at the level of the substrate supply and/or turn over. Expression of the two major factors involved in synthesis of P4, i.e., 3βHSD and STAR protein, was well investigated in canine CL during the course of dioestrus in both non-pregnant and pregnant animals (Kowalewski et al. 2009 and 2013). GnRH expression has been described in a wide range of non-pituitary normal tissues, as well as benign and malignant tumors derived from these tissues. tissues, such as ovary, placenta, oviduct, and testis express functional forms of GnRH–GnRH-R system in different species of animals: rat, rabbit, pig, cow, monkey, and human. Further, GnRH is believed to exert its directs effect(s) in an autocrine or paracrine manner eliciting a variety of responses depending on the type of target tissues and physiological conditions.

Background …the mechanisms of suppression and resumption of ovarian activity and, subsequently, luteal function after short/long- term inhibition of gonadal activity remains poorly understood. Despite recent progress in understanding canine luteal physiology during pregnancy and in non-pregnant cycles, To our knowledge, there is no information about the moleculer effects on the luteal function of the long term estrus suppression with GnRH agonists which is becoming increasingly popular for companion animals.

Aim to investigate the presence and expression levels of factors involved in luteal activity, as well as the expression of GnRH and GnRH-R in the canine luteal tissue following the first estrus after long-term prepubertal GnRH agonist-deslorelin treatment.

Material Pre-pubertal Deslorelin implant (Suprelorin®, 9,4 and 4,7 mg) treated; n=4 (supression period: 72.7 – 84.0 weeks, Kaya et al., 2015) Control (Placebo); n=4 Non-pregnant diestrus During luteal phase (5-65 d) at days: 5, 15, 25, 35, 45 and 65 after ovulation (n=3-5 per group) The animal material includes two different population. Totaly, eight healthy, crossbreed, female dogs were used. After the first sampling, deslorelin implant was inserted or sodium chloride 0.9% injected (controls, n=4) sc. in the inter-scapular region by using a single use applicator. To clarify the material-method section I would like to say that: We did the first part of this study as a clinical trial. It was a long term study and the results of the first part which includes clinical effects on the body development which was already published. After that, we examined the effects of deslorelin treatment on first observed estrus and we also operated these animals and tissue samples were collected for this study.

Methods Estrus cycles -vaginal cytology - blood sampling CL samples from deslorelin-treated and control bitches (OHE during the mid-luteal phase; days 30-45) CL samples from non-pregnant bitches throughout dioestrus (R-OHE) After the suppression period, estrus cycles were monitored every other day by vaginal cytology, serum P4 and E2 concentration. All animals were subject to ovariohysterectomy (OHE) during the mid-luteal phase (days 30-45) and mature corpora lutea (CL) samples were collected from deslorelin-treated and control bitches.

Methods Hormone analysis (ECLIA) - P4 and E2 Real-Time PCR - mRNA expression profiles GnRH-R, ERα, ERβ, PGR, PRLR, PTGES and PGE2 receptors (EP2 and EP4), VEGFA and VEGF receptors (VEGFR1 and -2), COX2, STAR protein and 3βHSD, Kiss1 and its receptor) Localization (GnRH-R) - in situ hybridization Pre-pubertal Non- pregnant diestrus

Results The mean length of the proestrus was 8.5±5.06 and 8.7±4.3 days in deslorelin-treated and in control bitches, respectively. Cytological examinations showed that there was no delay in the transition period from proestrus to estrus in treated group compared with the control.

Results Distribution of E2 and P4 P>0.05 This graph presents... No significant difference was observed for P4 and E2 concentrations between treated and control animals during proestrus and estrus period (P>0.05) P>0.05

Relative mRNA expression Results PTGES and EP2 expression in CL following first estrus P = 0.043 P = 0.200 * Relative mRNA expression **

Relative mRNA expression Relative mRNA expression Results ER alpha and ER beta expression in CL following first estrus P = 0.078 P = 0.198 Relative mRNA expression Relative mRNA expression Again, here you can see the graph

Relative mRNA expression Results PGR and StAR protein expression in CL following first estrus P = 0.095 Relative mRNA expression

Relative mRNA expression Results 3βHSD and COX2 expression in CL following first estrus P = 0.629 * Relative mRNA expression **

Relative mRNA expression Results Kiss1-R and PRL-R expression in CL following first estrus P = 0.343 P = 0.224 Relative mRNA expression Afterwards, we assessed

Relative mRNA expression Results GnRH-R expression in CL during diestrus. a ab ab Relative mRNA expression ab The graph shows the relative GnRH-R gene expression in CL from normal (non treated) dogs in the different time points of diestrus period. Although generally low, luteal GnRH-R expression was time-dependent (P<0.0001). The highest expression was detected at the beginning of the luteal phase, at days 5 and 15 p.o.. This was followed by a significant decrease towards the late luteal phase (day 5 vs. 65 p.o. P>0.001, and day 15 vs. 65 p.o. P<0.01) Afterwards, we b P<0.001

Results Localization of GnRH-R in CL during early diestrus in situ hybridization We were able to detected positive signals localized in the luteal cells

Results A similar GnRH-R expression pattern was observed for both treated and control animals. It was low or frequently below the detection limit. GnRH- gene expression determined in deslorelin treated and control group was n=1/4 and n=1/4, respectively (positive versus negative). Therefore, statistical calculations were not possible for these two parameters. P<0.001

Conclusion the results from our study indicate that long-term delay of puberty with GnRH analogue-deslorelin does not have carry- over-effects on subsequent estrus activity and luteal functionality in bitches. This is a productive area of research and may contribute to the development of new generation of GnRH analogues with highly selective and controlled action on different reproductive tissues and the target-specific GnRH analogues could be developed.

Thank you for your attention!..

Prof.Dr. Mariusz P. Kowalewski and I really appreciate Prof. Kowalewski and his great team for their support during my study... As a clinician, this study was my firts experience for molecular aspects... It was also a great opportunity to obtain a new scientific perspective and also to make new friends. Therefore... My sincere thanks to: Prof.Dr. Mariusz P. Kowalewski and colleagues from the Vetsuisse Faculty Zurich