Effect of kisspeptin on in vitro maturation of sheep oocytes

Aim: The aim of this study was to investigate the effect of kisspeptin (KP) on in vitro maturation (IVM) of sheep oocytes aspirated from the ovaries collected from slaughterhouse. Materials and Methods: Two different experiments were conducted to investigate the effect of KP (5, 10 and 15 µg/ml) alone (experiment 1) or in combination with follicle-stimulating hormone (FSH), luteinizing hormone (LH), and Estradiol (E2) (experiment 2) on IVM of sheep oocytes. Tissue culture medium 199 supplemented with Gentamicin was used as control medium. Good quality oocytes were randomly allocated into different IVM media and cultured at 38.5°C in 5% CO2 under humidified atmosphere for 24 h. The oocytes were evaluated for their cumulus cell expansion (CCE) and extrusion of the 1st polar body (PB) at the end of maturation. Results: The proportion of oocytes showing CCE and extrusion of PB was highest when the oocytes were matured in the medium supplemented with 10 µg/ml of KP. In experiment 2, oocytes were matured in 12 different maturation media (G1-G12: G1: Control, G2: KP alone, G3: FSH, G4: FSH+KP, G5: LH, G6: LH+KP, G7: E2, G8: E2+KP, G9: FSH+LH+E2, G10: FSH+LH+E2+KP, G11: FSH+LH+E2+fetal bovine serum (FBS), G12: FSH+LH+E2+FBS+KP). The proportion of oocytes showing cumulus expansion and PB extrusion was highest (98.33±1.05 and 89.17±2.38) when they were matured in FSH+LH+E2+FBS+KP (G12) and was significantly higher than other groups. The proportion of CCE and extrusion of PB was significantly increased when KP was supplemented to FSH and E2, but no effect was observed with LH. The maturation rates were significantly increased when FSH, LH, and E2 (G9) containing media were additionally supplemented with KP (G10). Conclusion: This study demonstrated that the addition of KP (10 µg/ml) to the FSH, LH, and E2 supplemented media would enhance the sheep oocyte maturation in vitro.


Introduction
Gonadotropin-releasing hormone (GnRH) has been known to play a major role in the control of reproduction for the last 30 years, but the mechanisms involved in the secretion of GnRH at cellular and molecular level are not completely understood [1]. Recent developments in reproduction with the use of kisspeptin (KP) have opened a new vista to understand these regulatory mechanisms of GnRH secretion [2]. KP seems to be involved in the onset of puberty, initiation of the breeding season and the dynamic changes of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion during estrous cycle [3]. KP, first identified as a metastasis suppressor molecule, is the product of the gene Kiss-1, which encodes a 145-amino acid peptide. It has biologically active peptides ranging from 10 to 54 amino acids in length [4]. Genes encoding KP (KISS1) and its receptors (KISS1R) have been documented in the ovaries of fish [5], hamsters [6], pigs and goats [7], primate, and human ovaries [8].
Administration of KP induces the secretion of gonadotropin hormones in many species such as pigs [9], bovines [10,11], ewes [12][13][14], goats [15], canines [16,17], and women [18,19]. The ability of KP to induce ovulation following intravenous administration has been demonstrated in prepubertal ewe lambs [20] and anestrous ewes [21]. The presence of high concentrations of KP in the porcine follicular fluid than in serum (335 vs. 25 pg/mL) and expression of KISS1Rs in oocytes and cumulus cells during in vitro maturation (IVM) pig oocytes supported the local involvement of KP on the development of oocytes within the follicle [22]. Apart from these in vivo studies, very few studies have also been conducted to see the effect of KP on IVM of oocytes. Supplementation of KP to maturation media has been reported to improve the efficiency of pig [22] and Therefore, taking into account the significance of KP in development of follicles, this study was undertaken to study the effect of KP on IVM of sheep oocytes.

Ethical approval
The experiments comply with guidelines laid down by the Institutional Ethical Committee.

Chemicals and media
All media, hormones, fetal bovine serum (FBS), and other chemicals were sourced from Sigma Chemical Co., USA, and plastic ware was from Nunc, Denmark, unless otherwise indicated. KP-10 (Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH 2 ) was purchased from Auropeptides, Hyderabad, India. HEPES buffered tissue culture medium 199 supplemented with 10% FBS (Handling medium) was used for washing and handling of oocytes. Heparin (25 IU/ml) was additionally added to the handling medium for collection of oocytes. Bicarbonate buffered tissue culture medium 199 (TCM199B) supplemented with gentamicin (50 µg/ml) was used as control medium for maturation of oocytes. The media used for transport and washing of ovaries, collection, handling, and maturation of oocytes were supplemented with gentamicin (50 µg/ml) and filter sterilized (0.22 µm) before use. Handling, collection, and maturation media were equilibrated with 5% carbon dioxide in air, in a humidified atmosphere at 38.5°C for at least 2 h before use.

Collection and IVM of oocytes
Collection and processing of ovaries for aspiration of cumulus oocyte complexes (COCs) were carried out as described by Arunakumari et al. [24,25]. Briefly, sheep ovaries were collected from a local slaughterhouse and transported to the laboratory in Dulbecco's phosphate buffered saline (D-PBS, Gibco, BRL, USA) maintained at 35°C, within 2 h after collection. Each ovary was trimmed off adjacent fat and ligaments. External surface of the each ovary was sterilized by rinsing once in 70% alcohol and thrice in D-PBS. COCs were aspirated aseptically from the visible follicles (>6 mm diameter) using a 20 G needle attached to 5 ml disposable syringe containing 2 ml of collection medium. COCs with homogeneous cytoplasm surrounded by at least three layers of compact cumulus cells were selected for IVM. The selected COCs were washed extensively in handling medium followed by respective IVM media. Subsequently, the 8-10 COCs were transferred into 50 µl droplets of different IVM media in 35 mm culture dishes. The droplets were overlaid with autoclaved pre-equilibrated mineral oil. The dishes with COCs were cultured in an incubator with 5% CO 2 under humidified air at 38.5°C for 24 h.

Evaluation of oocytes following IVM
At the end of IVM, the COCs were examined for cumulus cell expansion (CCE). The cumulus cells were removed by incubating them with 100 IU/ml of hyaluronidase for 15 min at 37°C and repeatedly passing them through a fire polished narrow bore glass capillary. The denuded oocytes were examined under an inverted microscope (TH4-200, Olympus, Japan) for the presence of the 1 st polar body (PB) in the perivitelline space.

Experimental design
Two separate experiments were designed. In the first experiment, influence of KP on IVM of the sheep COCs was investigated by supplementing the control medium with 5, 10 and 15 µg/ml of KP. After determining the best concentration of KP, the effect of KP with different hormones was investigated in the second experiment (Table-1).

Statistical analysis
The proportion of oocytes exhibiting CCE and extrusion of PB were analyzed by ANOVA followed by Duncan's multiple range test using SPSS software version 16.0 (SPSS Inc., USA) and presented as the mean±standard error.

Discussion
This study has demonstrated for the first time that addition of KP to FSH, LH, E 2 supplemented media can significantly increase the maturation rates of sheep oocytes in vitro. In experiment 1, TCM-199B was supplemented with 0, 5, 10, 15 µg/ml of KP, to identify the best concentration to support oocyte maturation in terms of CCE and extrusion of the 1 st PB. In sheep, 8-20 µg of KP was infused to monitor FSH and LH hormones in earlier studies [20,21]. Based on the CCE and extrusion of PB, 10 µg/ml of KP supported better for IVM of sheep oocytes. Similar to the present findings, maturation rate was improved when the bovine oocytes were matured in TCM-199 supplemented with 10 nM of KP (13 µg) [23]. In contrast, none of the pig oocytes extruded PB when maturation medium was supplemented with KP alone [22]. It is likely that the action of KP on cumulus cells and oocytes might be different from species to species. For example, in ovariectomized sheep, the increased LH response to KP administration was decreased by administration of KP antagonist (p234) [26], whereas in dogs it was not altered by KP antagonist (p271) [17]. In this study, the pattern of oocyte maturation significantly increased from 5 to 10 µg/ml and then decreased at 15 µg/ml. In vivo secretion of LH in response to KP administration was found to vary with dose, breed and physiological status of animal [13][14][15]27].
The action of KP on maturation of oocytes in vitro is different to that from in vivo. In vivo, KP directly acts on hypothalamic neurons and results in release of GnRH, followed by FSH and LH, which are responsible for follicle growth and ovulation. The expression of GnRHα and LH on cumulus cells and oocytes during IVM of pig oocytes suggested that KP activates its subordinate GnRHα, thereby increasing LH synthesis, which in turn will affect oocyte maturation [22]. In this study, to see the effect of KP with FSH, LH, and E 2 alone or in combination, the COCs were matured in different maturation media (experiment 2, Table-1). The addition of FSH, LH, and E 2 to IVM medium has been shown to improve the maturation rates in sheep oocytes, but the role of each hormone is not clear [28][29][30]. The proportion of  oocytes matured in medium supplemented with KP alone (G 2 ) was significantly lower than that of FSH, LH, and E 2 supplemented groups (G 3 , G 5 , G 7 , and G 9 ). In contrast, the bovine oocyte maturation rates in KP supplemented media was similar to that supplementing with FSH and FBS [23]. In this study, the addition of KP to FSH (G 4 ) and E 2 (G 8 ) supplemented media significantly enhanced extrusion of PB, compared to the media containing FSH or E 2 alone, whereas no improvement was observed in LH and KP combination (G 6 ) ( Table-3). It has been reported that priming of FSH is essential for the action of KP on IVM of pig oocytes. KP alone did not support extrusion of PB (0%), but in combination with FSH (76%) increased extrusion of PB [22]. Similarly, the addition of KP to the E 2 supplemented medium appears to suppress the negative effect of E 2 on nuclear maturation as shown in the pig [31].
The proportion of oocytes that showed CCE (95%) and extrusion of PB (80%) was significantly higher in standard FSH+LH+E 2 +FBS supplemented medium (G 11 ) than control and other combinations (G 2 -G 10 ). This might be due to the presence of FBS in the maturation medium which provides nutrients, vitamins, and growth factors to the cells surrounding the oocyte and therefore plays an important role in the maturation of oocytes [32]. The maturation rates in IVM medium were on par with the maturation of earlier studies in sheep [25,33,34]. However, the addition of KP to the FSH+LH+E 2 +FBS supplemented medium (G 12 ) significantly increased the CCE (98%) as well as PB extrusion (89%) when compared to FSH+LH+E 2 +FBS (G 11 ). The expression of KP receptors (KISS1Rs) in both oocytes and cumulus cells during IVM of pig oocytes and the increased maturation rates in FSH+KP supplemented media has been shown to be the result of the direct action of KP on oocytes and cumulus cells in an autocrine-paracrine fashion as in folliculogenesis [22]. Addition of various growth factors such as fibroblast growth factor-2, epidermal growth factor, insulin-like growth factor, insulin transferrin selenium, and growth hormone to IVM medium was also found to result in higher maturation and fertilization rates and improved embryo development in sheep oocytes [30,[35][36][37]. Further, the combinations of these growth factors and hormones successfully supported in vitro culture of sheep PFs and yielded fertilizable oocytes [24,38] and developed to morulae after IVM and fertilization [25], indicating the synergistic and direct action of hormones and growth factors on in vitro culture of sheep PFs and oocytes. The results of this study demonstrated the local interaction of KP with FSH, and E 2 during IVM of sheep oocytes as hypothesized in earlier studies [22] but it needs to be further explored by molecular approaches. The development competence of the oocytes matured in the presence of KP needs to be further evaluated by in vitro fertilization studies.

Conclusion
This study demonstrated that the addition of KP (10 µg/ml) to the FSH, E 2 , FSH+LH+E 2 supplemented media would enhance the sheep oocyte maturation in vitro. Addition of KP to FSH+LH+E 2 +FBS medium further increased the maturation rates in vitro.