SA1/SA2 cohesion proteins and SIRT1- NAD+ deacetylase modulate telomere homeostasis in cumulus cells and are eligible biomarkers of ovarian aging

Introduction

In the last years, many successfully experimental efforts have been
made with the aim of identifying molecular biomarkers predictive of
oocyte competence and to developing minimally invasive procedures
in the management of human reproduction (McKenzie et al., 2004).
The cumulus–oocyte complex (COC) has been recognized as a suit-
able target for this goal due to the bidirectional trafficking of metabolites, proteins, ions and regulative molecules (Assou et al., 2013). In addition, specific transcriptomic signatures in cumulus cells (CCs) appeared associated with pregnancy outcome (Assou et al., 2008).

Moreover, as indicated by clinical and experimental evidence, the relative telomere length in CCs at the time of oocyte collection may be a new candidate biomarker to discriminate mature reproductive cells with ontogenetic developmental potential. The telomeric termini of chromosomes are composed of extended tracts of short G-rich tan- dem repeat sequences, 5′-TTAGGG-3′, which are particularly prone to oxidative base damage (Saretzki and Von Zglinicki, 2002). The derivative 8-oxo dG lesions at G nucleotides are repaired less effi- ciently by OGG1 (8-oxoguanine DNA glycosylase) than non-telomere TG repeats, due to certain telomere configurations (Rhee et al., 2011). DNA damaging agents such as reactive oxygen species (ROS) affect telo- mere length and may trigger genomic instability, inefficient chromosomal pairing/segregation and anomalies of spindle apparatus in reproductive cells (Treff et al., 2011). Additionally, excessive telomere shortening has been linked to cellular senescence, aging and limited self-renewal of embryonic stem cells (Martens et al., 2000; Treff et al., 2011).

Recently, telomere length evaluation in CCs has proven to be suitable in predicting oocyte quality and embryo development in a large survey of COC samples from an IVF setting (Cheng et al., 2013). Telomere homeostasis is, however, a dynamic process under the influence of differ- ent molecular players which ensure specific biological functions and may contribute to set the telomere length (Martínez and Blasco, 2011; Keefe, 2016). Cohesins SA1/SA2 mediate sister chromatid cohesion at telo- mere termini (SA1) and along chromatid arms (SA2) (Canudas and Smith, 2009; Merkenschlager, 2010), while Sirtuin 1 (SIRT1), an NAD+- dependent protein deacetylase, senses DNA damage with the scope to preserve telomere integrity from oxidative stress (Carafa et al., 2012). We hypothesized that these cohesins may also modulate telomere homeostasis and thereby dictate oocyte ontogeny. In this study, we investigated SIRT1 and SA1/SA2 mRNA transcripts and DNA telomere sizing in CCs to uncover the contribution of these biomarkers to the physiology of the CCs and to assess whether definite clinical and biological parameters, routinely evaluated in patients undergoing assisted reproductive technologies, impact their expression profile.

Materials and Methods

Patient selection

Informed consent to participate to the study, approved by Local Ethic Committees, was obtained from women (n = 50) undergoing routine IVF/ ICSI treatment. All patients enrolled were unable to conceive naturally for at least one year before entering the study. The reasons for the infertility were: male factor infertility (n = 16), polycystic ovary syndrome (n = 6), tubal occlu- sion (n = 10) and endometriosis (n = 8) and unexplained (n = 10). The phys- ical and demographic characteristics of all recruited women are shown in Table I. Patients were randomly allocated to cohorts based on age (young < 34, n = 22 and old > 38, n = 24), BMI (<20kg/m2, n = 13; 20–25kg/m2, n = 25; >25 kg/m2, n = 12) and number of retrieved oocytes (<4, n = 28 and >6, n = 19). The laboratory investigations were carried out blindly in respect to the characteristics of the enrolled patients. Nicotinamide Mononucleotide

Ovulation induction

Ovarian stimulation was induced administering recombinant gonadotro- pins (Gonal-f, Pergoveris, Merck, Rome, Italy) at a dose of 150–300 IU per day from the first or second day of the menstrual cycle. The dose of gona- dotropins was adjusted according to ovarian response, as detected by ultrasound examination. As soon as the dominant follicle reached 14 mm in diameter, a gonadotropin-releasing hormone (GnRH) antagonist was administered daily, until the day of ovulation triggering which was obtained by HCG injection, when at least three follicles of size >16 mm were pre- sent in the ovaries. The oocyte retrieval was performed 34–36 h after the HCG injection. Follicular fluid was aspirated for COC recovery.

CCs isolation

CCs were isolated from COC and incubated in fertilization medium (Cook Medical, Limerick, Ireland). COCs were exposed to hyaluronidase 80IU (Irvine, USA); after 20–30s incubation, CCs were stripped from the oocyte with a micropipette 170 μm and 140 μm in diameter. CCs from fol- licles producing metaphase II oocytes were collected into pooled samples from each patient, then were washed two times pelleted and immediately transferred on ice before storage at −80°C until analysis.

Nucleic acids isolation from CCs

Genomic DNA and total RNA were extracted from pooled CCs of each patient by using the High Pure PCR Template Preparation Kit (Roche), according to the manufacturer’s instruction. This procedure allows the simultaneous elution of both DNA and RNA. Each sample was evaluated by spectrometry (Abs 260 nm/280 nm) and by QUBIT for quality control.

SA1/SA2 cohesion proteins and SIRT1- NAD+ deacetylase modulate telomere homeostasis in cumulus cells and are eligible biomarkers of ovarian aging

Introduction

In the last years, many successfully experimental efforts have been
made with the aim of identifying molecular biomarkers predictive of
oocyte competence and to developing minimally invasive procedures
in the management of human reproduction (McKenzie et al., 2004).
The cumulus–oocyte complex (COC) has been recognized as a suit-
able target for this goal due to the bidirectional trafficking of metabolites, proteins, ions and regulative molecules (Assou et al., 2013). In addition, specific transcriptomic signatures in cumulus cells (CCs) appeared associated with pregnancy outcome (Assou et al., 2008).

Moreover, as indicated by clinical and experimental evidence, the relative telomere length in CCs at the time of oocyte collection may be a new candidate biomarker to discriminate mature reproductive cells with ontogenetic developmental potential. The telomeric termini of chromosomes are composed of extended tracts of short G-rich tan- dem repeat sequences, 5′-TTAGGG-3′, which are particularly prone to oxidative base damage (Saretzki and Von Zglinicki, 2002). The derivative 8-oxo dG lesions at G nucleotides are repaired less effi- ciently by OGG1 (8-oxoguanine DNA glycosylase) than non-telomere TG repeats, due to certain telomere configurations (Rhee et al., 2011). DNA damaging agents such as reactive oxygen species (ROS) affect telo- mere length and may trigger genomic instability, inefficient chromosomal pairing/segregation and anomalies of spindle apparatus in reproductive cells (Treff et al., 2011). Additionally, excessive telomere shortening has been linked to cellular senescence, aging and limited self-renewal of embryonic stem cells (Martens et al., 2000; Treff et al., 2011).

Recently, telomere length evaluation in CCs has proven to be suitable in predicting oocyte quality and embryo development in a large survey of COC samples from an IVF setting (Cheng et al., 2013). Telomere homeostasis is, however, a dynamic process under the influence of differ- ent molecular players which ensure specific biological functions and may contribute to set the telomere length (Martínez and Blasco, 2011; Keefe, 2016). Cohesins SA1/SA2 mediate sister chromatid cohesion at telo- mere termini (SA1) and along chromatid arms (SA2) (Canudas and Smith, 2009; Merkenschlager, 2010), while Sirtuin 1 (SIRT1), an NAD+- dependent protein deacetylase, senses DNA damage with the scope to preserve telomere integrity from oxidative stress (Carafa et al., 2012). We hypothesized that these cohesins may also modulate telomere homeostasis and thereby dictate oocyte ontogeny. In this study, we investigated SIRT1 and SA1/SA2 mRNA transcripts and DNA telomere sizing in CCs to uncover the contribution of these biomarkers to the physiology of the CCs and to assess whether definite clinical and biological parameters, routinely evaluated in patients undergoing assisted reproductive technologies, impact their expression profile.

Materials and Methods

Patient selection

Informed consent to participate to the study, approved by Local Ethic Committees, was obtained from women (n = 50) undergoing routine IVF/ ICSI treatment. All patients enrolled were unable to conceive naturally for at least one year before entering the study. The reasons for the infertility were: male factor infertility (n = 16), polycystic ovary syndrome (n = 6), tubal occlu- sion (n = 10) and endometriosis (n = 8) and unexplained (n = 10). The phys- ical and demographic characteristics of all recruited women are shown in Table I. Patients were randomly allocated to cohorts based on age (young < 34, n = 22 and old > 38, n = 24), BMI (<20kg/m2, n = 13; 20–25kg/m2, n = 25; >25 kg/m2, n = 12) and number of retrieved oocytes (<4, n = 28 and >6, n = 19). The laboratory investigations were carried out blindly in respect to the characteristics of the enrolled patients. Nicotinamide Mononucleotide

Ovulation induction

Ovarian stimulation was induced administering recombinant gonadotro- pins (Gonal-f, Pergoveris, Merck, Rome, Italy) at a dose of 150–300 IU per day from the first or second day of the menstrual cycle. The dose of gona- dotropins was adjusted according to ovarian response, as detected by ultrasound examination. As soon as the dominant follicle reached 14 mm in diameter, a gonadotropin-releasing hormone (GnRH) antagonist was administered daily, until the day of ovulation triggering which was obtained by HCG injection, when at least three follicles of size >16 mm were pre- sent in the ovaries. The oocyte retrieval was performed 34–36 h after the HCG injection. Follicular fluid was aspirated for COC recovery.

CCs isolation

CCs were isolated from COC and incubated in fertilization medium (Cook Medical, Limerick, Ireland). COCs were exposed to hyaluronidase 80IU (Irvine, USA); after 20–30s incubation, CCs were stripped from the oocyte with a micropipette 170 μm and 140 μm in diameter. CCs from fol- licles producing metaphase II oocytes were collected into pooled samples from each patient, then were washed two times pelleted and immediately transferred on ice before storage at −80°C until analysis.

Nucleic acids isolation from CCs

Genomic DNA and total RNA were extracted from pooled CCs of each patient by using the High Pure PCR Template Preparation Kit (Roche), according to the manufacturer’s instruction. This procedure allows the simultaneous elution of both DNA and RNA. Each sample was evaluated by spectrometry (Abs 260 nm/280 nm) and by QUBIT for quality control.