Effect of Kisspeptin on the Developmental Competence and Early Transcript Expression in Porcine Oocytes Parthenogenetically Activated with Different Methods

Recent studies showed the modulatory effect of kisspeptin (KP) on calcium waves through the cell membrane and inside the cell. Spermatozoon can induce similar ooplasmic calcium oscillations at fertilization to trigger meiosis II. Here, we evaluated the effect of KP supplementation with 6-dimethylaminopurine (6-DMAP) for 4 h on embryonic development after oocyte activation with single electric pulse, 5 µM ionomycin, or 8% ethanol. Compared to control nonsupplemented groups, KP significantly improved embryo developmental competence electric- and ethanol-activated oocytes in terms of cleavage (75.3% and 58.6% versus 64% and 48%, respectively, p < 0.05) and blastocyst development (31.3% and 10% versus 19.3% and 4%, respectively, p < 0.05). MOS expression was increased in electrically activated oocytes in presence of KP while it significantly reduced CCNB1 expression. In ionomycin treated group, both MOS and CCNB1 showed significant increase with no difference between KP and control groups. In ethanol-treated group, KP significantly reduced CCNB1 but no effect was observed on MOS expression. The early alterations in MOS and CCNB1 mRNA transcripts caused by KP may explain the significant differences in the developmental competence between the experimental groups. Kisspeptin supplementation may be adopted in protocols for porcine oocyte activation through electric current and ethanol to improve embryonic developmental competence.


Introduction
In mammals, meiosis II resumes in mature oocytes after fertilization and is mainly mediated by the highly repetitive calcium (Ca 2+ ) signals that last for 2-3 h rather than a single Ca 2+ rise [1,2]. Ca 2+ modulates the transition of the cell from one meiotic phase and cell cycle control checkpoints to the following phase till the extrusion of the second polar body [3]. Lack of intracellular Ca 2+ elevation prevents spontaneous meiosis resumption in vitro [4]. Moreover, injection of Ca 2+ in mouse oocytes has been shown to cause parthenogenetic activation and subsequent embryonic development [1].
The c-Mos (MOS) proto-oncogene is a serine/threonine kinase expressed in vertebrate oocytes and plays a crucial role in meiosis and germ cell development [5,6]. MOS is functionally expressed during G2/M progression of oocyte cell cycle [7] and was found to be expressed highly in oocytes while it reduced with the cleavage and mitosis of the zygote [8]. MOS activates mitogen-activated protein kinase (MAPK), which in turn activates M-phase promoting factor 2 BioMed Research International (MPF) [6]. MPF is a protein complex comprised of a catalytic subunit p34 cdc2 with serine-threonine kinase activity and a regulatory subunit cyclin B (CCNB1). MPF is regulated by the binding of CCNB1 with cyclin-dependent kinase 1 (Cdc2) and phosphorylation of threonine 161 and dephosphorylation of tyrosine 15 and threonine 14 [9]. Notably, there is a regulatory interplay between Ca +2 , MOS, and CCNB1 in a feedback manner to regulate the exit from meiosis II [10][11][12][13][14][15].
Parthenogenetic activation of porcine oocytes has been carried out using different methods such as electrical stimulus, chemical activation, and ethanol treatment [16][17][18][19]; however, the underlying molecular mechanism is questionable. During parthenogenesis protocols, activation is usually followed by the inhibition of protein phosphorylation with 6dimethylaminopurine (6-DMAP), a protein kinase inhibitor, resulting in efficient oocyte activation and development of parthenotes into the blastocyst stage [20]. 6-DMAP inhibits MPF reactivation, thereby triggering a kinetic similar to that occurring after fertilization [21,22]. However, interfering with one or several kinases involved in other cellular functions may alter the subsequent cellular events.
Interestingly, cyclin degradation is activated by fertilization; however this activation is transitory since cyclin B1 levels recover between Ca 2+ oscillation spikes. Therefore, continued cyclin degradation at basal Ca 2+ levels requires multiple spikes to complete meiosis II [23].
Therefore, this study aimed to examine whether KP, as a Ca 2+ modulator, can differentially support the transition of oocytes from MII stage to further embryonic developmental stages when oocytes activated with electric stimulus, ionomycin, or ethanol through investigating the effect of KP supplementation on the expression of early maternal transcripts involved in meiotic progression, MOS and CCNB1.

Parthenogenetic Activation and KP Supplementation.
After 44 h of IVM, oocytes and cumulus cells were separated by pipetting with 0.1% hyaluronidase in Dulbecco's phosphate-buffered saline (PBS) supplemented with 0.1% polyvinyl alcohol. Denuded oocytes were randomly separated into three groups and subjected to one of the following parthenogenetic activation methods: (1) in electrical activation, oocytes were activated with a single DC pulse of 1.5 kV/cm for 100 s in mannitol (0.25 M) using BTX Electro Cell Manipulator 2001 designed electrodes chamber (BTX, Inc., San Diego, CA, USA); (2) in ionomycin activation, oocytes were incubated in TCM-199 supplemented with 10% fetal bovine serum (FBS) and 5 M ionomycin for 5 min in a dark chamber; or (3) in ethanol activation, oocytes were incubated in TCM-199 containing 10% FBS and 8% ethanol for 10 min. Activated oocytes were washed in TCM and cultured in 500 L of 4 mM 6-DMAP with or without 1 M KP for 4 h at 39 ∘ C in an atmosphere of 5% CO 2 , 5% O 2 , and 90% N 2 to produce diploid zygotes.

Experimental Design.
Denuded oocytes were randomly allocated into three groups, 75 oocytes each, and incubated in 50 L microdrops of TCM-199 supplemented with 10% fetal bovine serum (FBS) for 10 min. Oocytes were then activated with specific treatment (electric, ionomycin, or ethanol activation), and then, a pool of oocytes ( = 25) was immediately collected from each group to extract mRNA (0 h after activation). After incubation with 6-DMAP with or without KP, another pool of oocytes ( = 25) was collected to extract mRNA from each group (4 h after activation batch). The remaining 25 oocytes were then used for in vitro culture. The experiment was repeated six times and the data were presented as mean ± SEM.

In Vitro Culture of Embryos.
Presumptive zygotes were washed and cultured in 30 L microdrops of porcine zygote medium-5 covered with mineral oil at 39 ∘ C in presence of 5% CO 2 , 5% O 2 , and 90% N 2 . Embryos were evaluated

Results and Discussion
Results showed that supplementation of 6-DMAP culture with KP (KP+) significantly increased embryo cleavage proportion in oocytes activated with electric pulse and ethanol (75.42 ± 2.9% and 58.0 ± 0.6%, respectively) as compared with the control groups (KP−) ( Table 2) with maximum effect observed with electrical activation ( < 0.05). On the other hand, KP supplementation had no advantageous effect on the oocytes activated with ionomycin (Table 2, Figure 1). Similar trend was observed for blastocysts; KP-supplemented groups activated with electric pulse and ethanol showed a significant increase in blastocyst proportion (31.75 ± 0.9% and 10.25 ± 0.25%, respectively) as compared with control groups ( Table 2, < 0.05). On the other hand, KP supplementation showed no effect on blastocyst formation in oocytes activated with ionomycin.
Additionally, a significant increase in embryo cleavage and blastocyst was observed following oocyte activation with electric pulse (64.25 ± 2.1% and 19.5 ± 0.3%, respectively) and ionomycin (63.25 ± 0.25% and 22.25 ± 0.8%, respectively) as compared to that in ethanol-activated oocytes (47.5 ± 0.3% and 4.0 ± 0.6%, respectively, < 0.05) (Table 2, Figure 1). This is consistent with the results by Cheng et al. [16], wherein the electrical and ionomycin activation resulted in a threefold increase in developmental competence of porcine oocytes as compared with the ethanol-mediated activation. Moreover, ethanol treatment in oocytes was reported to yield a very low percentage of cleavage and blastocyst formation [19]. On the contrary, Koo et al. [41] showed that electrical activation significantly increased the cleavage and blastocyst development as compared with ionomycin activation.
Recent reports have described the mechanism of action of KP through the modulation of Ca 2+ oscillation in different cell types, including spermatozoa [32][33][34][35][36]. In addition, KP is known to cause biphasic increase in intracellular Ca 2+ characterized by a short and transient phase followed by a  sustained phase [37]. Following oocyte activation, the oscillation in Ca 2+ signals, in particular the early Ca 2+ /calmodulindependent kinase pathways, results in the emission of the second polar body [42]. The recruitment of maternal mRNAs is thought to be a graded response to the number of Ca 2+ oscillations experienced by the oocyte [43]. In addition, artificial methods of oocyte activation may result in the recruitment of the maternal mRNA in the absence of Ca 2+ elevation [44], indicative of the indirect effect of Ca 2+ on the process downstream of cell cycle resumption. Electric pulse showed a higher and longer Ca 2+ transient in the oocyte immediately after its activation [45]. Moreover, electrical activation induces a rapid reduction in histone H1 kinase activity [46]. However, the elevation in intracellular levels of free Ca 2+ by ionomycin was less effective for parthenogenetic activation of oocytes than that caused by electrical pulse [47]. Studies have reviewed the different mechanistic pathways for porcine parthenogenesis [48,49]. In particular, electrical pulse causes influx of Ca 2+ by increasing the permeability of the oolemma [50,51], while 7% ethanol and calcium ionophores cause a surge in the intracellular Ca 2+ from Ca 2+ stores by increasing the intracellular pH [47,[52][53][54]. The sodium-hydrogen (Na + /H + ) antiporter and the bicarbonatechloride (HCO 3 − /Cl − ) exchanger are suggested to play no role during calcium ionophore treatment; however, ethanol treatment in oocytes may be dependent upon either Na + or HCO 3 − flux (or both) [53]. On the other hand, calcium ionophores inhibit the vacuolar-type H + ATPases, thereby increasing the intracellular pH [53]. The pH rise plays an important role in activating oocyte metabolism and increasing DNA and protein synthesis [55]. Since the Ca +2 ions have two opposing roles during Xenopus oocyte maturation, it negatively regulates meiosis entry by delaying the activation of the cell cycle machinery (downstream of protein kinase A and upstream of MOS), while on the other hand, it is required for completion of meiosis [56]. Therefore, modulation of temporal calcium influx to the ooplasm is essential for successful meiosis. The three activation methods use calcium signals as the main pathway for oocyte activation; however, how acute or repetitive calcium influx is and for how long this influx continues are questions for clarifying the difference between these methods for supporting meiosis II and early embryo development.
We investigated the possible cause of the difference between activation methods with or without KP supplementation. MOS and CCNB1 transcripts were relatively quantified through real-time PCR immediately after activation (0 h after activation), and 4 h after activation and culture in 6-DMAP (4 h after activation) with or without KP. Statistical analysis showed significant interaction among the treatments and the temporal expression of MOS and CCNB1.
It has been clarified that 6-DMAP, a phosphatase inhibitor, caused a higher and longer Ca 2+ transient and may activate oocytes by changing Ca 2+ oscillations in oocytes and inhibiting phosphotyrosine dephosphorylation after parthenogenetic activation [45]. However, oocytes activated with calcium ionophores and 6-DMAP displayed abnormal pattern of karyokinesis during the first cell cycle [57].
Although 6-DMAP-mediated inhibition of protein kinase is an efficient way to induce oocyte activation, interfering with one or several kinases involved in other functions may be deleterious for cellular events after activation. Therefore, we aimed to examine whether KP may act as a Ca 2+ modulator during this period.
Kisspeptin supplementation after 4 h from activation resulted in differential alteration in the expression patterns of MOS and CCNB1 in different treatment groups; it significantly increased MOS expression (2.5-fold, Table 2) when compared with both control (KP−) and 0 h after activation in electrically activated oocytes ( < 0.05; Figures 2(a) and  2(b)). In ethanol-treated group, KP significantly decreased the level of CCNB1 by 0.3-fold when compared with 0 h after activation and control nonsupplemented group ( < 0.05); however, no effect was observed on MOS (Figures 2(e) and 2(f)). On the other hand, oocytes treated with ionomycin showed no difference on MOS and CCNB1 both with and without KP supplementation ( = 0.9), although MOS and CCNB1 expressions significantly increased (∼2.5-fold) after 4 h from activation (Figures 2(c) and 2(d), < 0.05).
Some reports showed that mRNA expression of protooncogenes (such as MYC) is tightly and coordinately regulated and rapidly turned over with half-lives of 9-40 min [23,58,59]. Moreover, transcription rates for transiently expressed genes and mRNA stability are often rapidly and coordinately affected by calcium-dependent signaling pathways [60,61]. These observations might explain the rapid transcriptional changes in proto-oncogene MOS and CCNB1 associated with oocyte stimulation and its incubation with calcium modulator, KP.
The early changes in cyclin B and c-Mos complexes are critical for pronuclear formation; after oocyte fertilization or parthenogenetic activation, an increase in intracellular Ca 2+ causes degradation of c-Mos protein, inactivation of cytostatic factor (CSF) and MPF, and release of eggs from meiotic metaphase arrest [62]. These results suggest that the early changes in MOS and CCNB1 may influence the embryonic developmental competence, including cleavage and subsequent mitosis stages until the blastocyst formation. On the other hand, Lazar et al. [63] clarified that MOS mRNA expression is associated with meiosis progression and occurs independent of MAPK. In addition, Koo et al. [41] showed inactivation of MPF and MAPK after oocyte activation with single electrical stimulus.
In the current study, KP increased the level of MOS (2.5fold) in electrically activated oocytes, highlighting the need of MOS for successful completion of cell cycle progression after activation [6,7]. Moreover, supplementation of the electrically or ethanol-activated oocytes with KP significantly reduced the level of CCNB1 transcript, which is essential for MPF inhibition to allow meiosis II to proceed [9]. Interestingly, the interplay between MOS and CCNB1 plays an important role during meiosis in mammalian oocytes [10][11][12][13][14][15]. Physiologically, a spermatozoa Ca 2+ signal induces degradation of cyclin B1 which in turn results in exit of oocyte from metaphase II arrest and permits extruding the second polar body [14]. On the other hand, MOS is critical for protracted metaphase II arrest, but it is not required for its establishment [64,65]. Paradoxically, MPF activity may negatively regulate the MOS pathway; Yamamoto et al. [66] showed that when MPF activity reaches a critical lower level, the c-Mos/MAPK pathway suppresses cyclin B degradation in order to elevate MPF levels, while elevation of MPF beyond a critical upper level activates cyclin B degradation.

Conclusion
Kisspeptin supplementation with 6-DMAP culture improved the developmental competence of oocytes activated with electric pulse and 8% ethanol. Relative quantitative analysis of the transcript showed changes associated with the efficient BioMed Research International 7 activation of oocytes; MOS expression was increased after electric and ionomycin activation and this effect was magnified in presence of KP in electrically activated oocytes. This could explain the difference in cleavage and blastocyst development observed in the two groups as compared with the ethanol-activated group. On the other hand, CCNB1 expression was decreased in electric and ethanol-activated oocytes treated with KP. This result might also explain the interplay between different regulatory components of calcium ions, MOS/CCNB1/MAPK pathway to exit from oocyte metaphase II arrest which in turn reflects difference in embryo cleavage, and blastocyst development observed between groups treated with the calcium modulatory peptide KP.
We suggest that KP supplementation may be adopted in the protocol for electric and ethanol activation of porcine oocyte to improve the embryonic developmental competence.

Additional Points
Availability of Data and Material. All data generated or analyzed during this study are included in this article. Any inquiries should be sent directed to the corresponding author.

Conflicts of Interest
The authors declare that they have no conflicts of interest.