Journal of Genetic Disorders & Genetic Reports ISSN: 2327-5790

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Research Article, J Genet Disor Genet Rep Vol: 6 Issue: 1

PROK2 and PNCK – Novel Proteins Present in Follicular Fluid of Mature and Immature Human Oocytes: Preliminary Study

Burnik Papler T1, Verdenik I2, Devjak R3 and Vrtacnik Bokal E1*
1Department of Human Reproduction, Division of Gynecology, University Medical Center Ljubljana, Slovenia
2Division of Gynecology, University Medical Center Ljubljana, Slovenia
3Institute of Oncology, Ljubljana, Slovenia
Corresponding author : Prof. E. Vrtacnik Bokal, MD, PhD
Department of Human Reproduction, Division of Gynecology, University Medical Center Ljubljana, Slajmerjeva 3, 1000 Ljubljana, Slovenia
Tel: +38115226228
E-mail: [email protected]
Received: January 12, 2017 Accepted: February 01, 2017 Published: February 07, 2017
Citation: Burnik Papler T, Verdenik I, Devjak R, Vrtacnik Bokal E (2017) PROK2 and PNCK – Novel Proteins Present in Follicular Fluid of Mature and Immature Human Oocytes: Preliminary Study. J Genet Disor Genet Rep 6:1. doi: 10.4172/2327-5790.1000150

Abstract

Analysis of follicular fluid (FF), granulosa (GC) and cumulus cells (CC) gene expression can unravel the physiology of oocyte maturation. In our previous study, a meta-analysis of microarray results showed that prokineticin 2 (PROK2) and pregnancy up-regulated non-ubiquitis CaMkinase (PNCK) genes are differentially expressed between GC and CC. Follicular fluid represents an important medium for oocyte maturation. It contains mediators of folliculogenesis and their identification would help us better understand the process of oocyte maturation.  The aim of this preliminary study was to determine whether PROK2 and PNCK genes are translated into proteins in FF. Furthermore, we wanted to determine whether their levels differ between FF of mature and immature oocytes. Thirty-six women included in the IVF processes for tubal factor of infertility were included in the study. Short GnRH antagonist prostocol was used for ovarian stimulation. Individual follicles were aspirated with transvaginal puncture. Enzyme-linked immunosorbent assay (ELISA) was used for determination of PROK2 and PNCK proteins in individual follicular fluid samples. Both proteins were present in FF of mature and immature oocytes. Level of PNCK was significantly higher in FF of mature (0.63 µg/L ± 1.78 µg/L) as compared to immature (0.17 µg/L ± 0.33 µg/L) oocytes. Level of PROK2 did not differ between mature and immature oocytes. Significant positive correlation between PROK2 and PNCK levels existed in FF of mature oocytes. In this preliminary study, we confirmed the presence of two novel proteins in FF of mature and immature human oocytes. Further studies with larger number of samples are needed to determine whether the concentration of PNCK truly differs between mature and immature oocytes. Also, their function in the process of oocyte maturation remains to be determined.

Keywords: Follicular fluid; Proteins; PROK2; PNCK; Oocyte maturation

Keywords

Follicular fluid; Proteins; PROK2; PNCK; Oocyte maturation

Abbreviations

BMI: Body Mass Index; CC: Cumulus Sells; COC: Cumulus-Oocyte Complex; ELISA: Enzyme-Linked Immuno Sorbent Assay; FF: Follicular Fluid; GC: Granulosa Cells; hCG: Human Chorionic Gonadotrophin

Background

The process of human ovarian follicle development and oocyte maturation is complex, and factors involved in it remain poorely known. During folliculogenesis, somatic cells develop into two distinct types, granulosa cells (GC), which compose the follicular wall, and cumulus cells (CC), which surround the oocyte. Bidirectional communication between oocyte and somatic cells in the follicular microenvironment is necessary during the process of oocyte maturation [1]. During folliculogenesis, antrum filled with follicular fluid (FF) develops. It contains plasma proteins and proteins produced by somatic follicular cells [2].
Follicular fluid serves as a medium for transport of paracrine and autocrine signaling mediators that are necessary for the development of a mature oocyte.
Identification of substances that are crucial for follicular development and oocyte maturation will help optimize the process of in vitro maturation of immature oocytes and provide objective biomarkers of oocyte quality. It is believed that because of the intimate connection between oocyte, FF, GC and CC analysis of these biologic substances has the potential to discover such biomarkers and unravel the physiology of folliculogenesis.
Several studies have tried to improve our understanding of the process of oocyte maturation by analyzing gene expression in human GC and CC [3,4]. Analysis of FF content has also been used for determination of oocyte maturity and as such, determination of substances that take part in the process of oocyte maturation [5].
In our previous study [6], we identified prokineticin 2 (PROK2) and pregnancy up-regulated non-ubiquitis CaMkinase (PNCK) genes were differentially expressed between GC and CC by using microarray analysis. PROK2 expression was higher in GC whereas PNCK expression was higher in CC. To the best of our knowledge, our study was the first to describe these two genes are expressed in human GC and CC. For this reason, their potential role in human folliculogenesis and oocyte maturation is unknown.
The aim of the present study was to determine, whether PROK2 and PNCK proteins are present in human FF. Furthermore, we wanted to determine, whether their concentration differs between FF of mature, metaphase II (MII), and immature, metaphase I (MI), oocytes.

Methods

Thirty-six (36) IVF patients were included in this study. The inclusion criteria were: age less than 35 years, body mass index (BMI) between 17 and 26 kg/m2, tubal cause of infertility, first or second IVF procedure and normal partner’s spermiogram according to the WHO criteria [7]. The study was approved by the National Medical Ethics Committee of the Republic of Slovenia. All participants signed an informed consent prior to inclusion in the study.
All patients were treated with recombinant FSH (Puregon; Schering Plough, New Jersey, USA) and GnRH antagonist cetrorelix acetate (Cetrotide; AstaMedica AG, Frankfurt, Germany). Vaginal ultrasound examination was used to monitor follicular development. Final follicular maturation was induced by administering 10,000 IU of human chorionic gonadotrophin (hCG) (Pregnyl; N.V. Organon, Oss, the Netherlands) when at least three follicles were ≥17 mm. Ultrasound guided transvaginal puncture was performed 34-36 h later. Each follicle was aspirated separately. After that, cumulusoocyte complexes (COC) were removed from follicular fluid and oocyte maturity was assessed. Classical IVF was used for fertilization of mature, MII, oocytes. Embryo transfer was performed on day 5 after oocyte retrieval. Immature, MI, oocytes were discarded.
After aspiration and COC removal, individual FF samples were centrifuged (300 × g for 10 min). The supernatant was snap frozen in liquid nitrogen and stored at 80°C until analysis. Only FF containing one mature or immature oocyte were stored. Fifty-three individual FF samples, 19 containing immature, MI, and 34 containing mature, MII, oocytes, were analyzed. Enzyme-Linked ImmunoSorbent Assay (ELISA) kits for Prokineticin 2 and Pregnancy Up Regulated Non - Ubiquitously Expressed CaM Kinase (Wuhan USCN Business Co., Ltd.) were used according to manufacturer’s protocol.

Statistical Analysis

Data were analyzed using IBM SPSS Statistics 21.0. Mann- Whitney-Wilcox test was used for comparison of levels of proteins between mature and immature oocytes. A Spearman rank order correlation was used to assess relationship between both proteins separately in each group. A P value of <0.05 was considered statistically significant.

Results

Patient’s and IVF characteristics are represented in Table 1.
Table 1: Patient and cycle characteristics.
Both of the examined proteins were present in FF containing mature as well as immature oocytes. Level of PROK2 was 0.68 μg/L ± 1.02 μg/L and 0.69 μg/L ± 0.81 μg/L for FF of immature and mature oocytes, respectively. Level of PNCK was 0.17 μg/L ± 0.33 μg/L in FF of immature oocytes and 0.63 μg/L ± 1.78 μg/L in FF of mature oocytes (Table 2). There is no significant difference in PROK2 value between mature and immature oocytes. PNCK value is significantly higher in FF containing mature oocytes. Significant positive correlation exists between PROK2 and PNCK levels in FF of mature oocytes (Table 3).
Table 2: Level of PROK2 and PNCK proteins in FF according to oocyte maturity.
Table 3: Spearman’s correlation coefficient between PROK2 and PNCK level in FF of mature oocytes.

Discussion

We analyzed FF of immature and mature oocytes to determine the presence of two proteins, PROK2 and PNCK, in the present study. Results have shown that both of these proteins are present in FF containing mature as well as immature oocytes. Furthermore, PNCK protein is significantly more abundant in FF derived from follicles containing mature, MII, oocytes.
In our previous study [6], we performed a meta-analysis of gene expression differences between human GC and CC and for the first time, determined novel genes, PROK2 and PNCK, are expressed in these cells. Genetic information is translated into proteins through mRNA expression and proteins are directly involved in life processes. It is important to verify mRNA expression by determining protein expression, as it is known that almost 50 % of mRNA is not translated or it is partially degraded before protein synthesis [8]. Post-translational modifications occurring within cells are mainly responsible for the discrepancies noted between the genome and the expressed proteome [9]. Proteins are the ones that dictate cellular functions. The present study confirmed that PROK2 and PNCK genes, expressed in GC and CC, are translated into proteins and present in FF of mature and immature oocytes.
PROK2 is a protein that exerts its effects through G-protein coupled PROKR2 receptor [10]. It is involved in the control of circadian rhythms [11], sexual maturation [12] and angiogenesis [13]. Defects of PROK2 pathway in humans cause hypogonadotropic hypogonadism due to the reduction of GnRH neurons in the preoptic hypothalamic region, which causes GnRH deficiency [14]. Two to 7 % of patients with Kallmann syndrome who suffer from hypogonadotropic hypogonadism have inactivating mutations of PROK2 and PROKR2 [15]. Possible functional role of PROK2 protein in human folliculogenesis has not yet been determined. In the present study, there were no differences in PROK2 protein levels between FF of mature and immature oocytes. In our previous study [6], PROK2 gene was connected to tumor necrosis factor (TNF) gene that causes apoptosis and breakdown of the extracellular matrix in the follicle wall during mammalian ovulation [16]. Degradation of the basement membrane surrounding GC and the underlying ovarian surface epithelium is required for release of the cumulus – oocyte complex at the time of ovulation. One possible explanation why there were no differences in PROK2 protein levels between FF of mature and immature oocytes is that oocyte pick-up in IVF cycles is performed before follicle rupture, that is, before degradation of the follicle wall. The other possible explanation is that PROK2 protein is produced by GC and its main function is on the ‘outer’ side of the follicle and not in the FF.
The level of PNCK protein was significantly higher in FF containing mature oocytes in the present study. Function of this protein in human folliculogenesis has not yet been established. It is known that over-expression of PNCK causes epidermal-growth factor receptor (EGFR) protein degradation, which leads to inhibition of EGF – induced mitogen-activated protein kinase (MAPK) activation [17]. Activation of Ras-MAPK3/1 pathway in GC and CC is necessary for CC expansion, final oocyte maturation and GC luteinization and this pathway is activated with EGF -like factors that act on EGFR [18]. We presume that PNCK protein maintains inactivation of Ras- MAPK3/1 pathway, once the process of oocyte maturation is finished. Results of the present study support this assumption, as the level of PNCK protein is higher in FF of mature oocytes.
Significant positive correlation was seen between PROK2 and PNCK levels in FF of mature oocytes but not in immature oocytes. It seems that levels of these two proteins are inter-connected during the final stages of oocyte maturation. This finding again confirms the assumption that PROK2 has a function during the final stages of folliculogenesis.
The limitation of our study is a relatively small sample size. Therefore, further studies, containing larger number of FF are needed to reliably confirm significant differences in PNCK concentrations between mature and immature oocytes. Nevertheless, we have confirmed the translation of PROK2 and PNCK genes into proteins that are present in human FF.

Conclusions

To the best of our knowledge, PROK2 and PNCK proteins have not yet been described to be present in human FF. Analysis of human FF components has the potential to improve our knowledge of the physiological processes controlling folliculogenesis and oocyte maturation.Further studies are needed to establish the role of these proteins during oocyte maturation/folliculogenesis.

Ethics Approval and Consent to Participate

Slovenian National Ethics committee approved the study. All participants signed an informed consent prior to participation in the study.

Availability of Data and Materials

The data analysed during the current study available from the corresponding author on reasonable request.

Competing Interests

The authors declare they have no competing interests whatsoever.

Funding

The study was funded by the Slovenian Research Agency (https://www.arrs. gov.si/en/novo.asp), grant number P3-0326. Tanja Burnik Papler is funded as a young researcher by the Slovenian Research Agency. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author’s Contributions

TBP and EVB designed the study and collected the materials. TBP performed experiments and wrote the manuscript. IV performed statistical analyses. All authors revised and approved the final version of the manuscript.

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