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BOR - Papers in Press, published online ahead of print June 15, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.041103
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BIOLOGY OF REPRODUCTION 73, 737–744 (2005)
DOI: 10.1095/biolreprod.105.041103
© 2005 by the Society for the Study of Reproduction, Inc.

Maturation of Bovine Oocytes in the Presence of Leptin Improves Development and Reduces Apoptosis of In Vitro-Produced Blastocysts1

Marc Boelhauve 3, Fred Sinowatz 4, Eckhard Wolf 2, 3, and Fabíola F. Paula-Lopes 3

Department of Molecular Animal Breeding and Biotechnology,3 Ludwig-Maximilian University, 85764 Oberschleissheim, Germany Institute of Veterinary Anatomy, Histology, and Embryology,4 Ludwig-Maximilian University, 80539 Munich, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The series of events associated with oocyte growth and maturation determines the oocyte's ability to undergo successful fertilization, cleavage and embryonic development. Among the molecules involved in these events, leptin has been identified as a modulator of oocyte function. Experiments were conducted to determine whether leptin treatment of oocytes during maturation affects their developmental capacity after fertilization and whether it has long-lasting effects on apoptosis and gene expression in the resulting blastocysts. Cumulus-oocyte complexes (COCs) were matured in serum-free medium containing 0 (control), 1, 10, or 100 ng/ml leptin or in medium supplemented with 10% (v/v) estrous cow serum (ECS). Addition of leptin during oocyte maturation had no effect on cleavage rate after fertilization. However, an increased proportion of oocytes that matured in the presence of 1 or 10 ng/ml leptin developed to blastocysts, which exhibited increased cell numbers. The proportion of apoptotic cells was reduced in blastocysts originating from leptin- or ECS-treated oocytes. Transcript levels of the genes encoding leptin receptor (LEPR), signal transducer and activator of transcription (STAT3), BCL2 associated X-protein (BAX), and baculoviral inhibitor of apoptosis protein repeat-containing 4 (BIRC4, also known as XIAP), were determined by reverse transcriptase-quantitative polymerase chain reaction analysis of expanded and hatched blastocysts. Depending on the dose used, leptin treatment of oocytes resulted in increased LEPR, STAT3, and BIRC4 mRNA levels and reduced BAX mRNA levels in blastocysts. In conclusion, leptin improved the ability of the oocyte to sustain embryonic development and had long-term effects on blastocyst apoptosis and transcript abundance of LEPR, STAT3, and apoptosis-associated genes.

apoptosis, embryo, leptin, leptin receptor, oocyte development


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leptin, the 16-kDa product of the leptin gene (LEP), is a pleiotropic peptide secreted primarily by adipocytes. Even though leptin was initially identified as a signal that regulates food intake and energy expenditure [1, 2], it has also been implicated in a wider range of physiological functions, including reproduction [3–5]. The first evidence that leptin was required for reproduction came from studies in mice. Lepob/Lepob mutant mice, which express a truncated leptin protein, are obese and infertile [2, 6]. Fertility of Lepob/ Lepob mice can be restored by exogenous leptin supplementation. Recently, the leptin system has been localized in the reproductive tract of several species. Leptin has been detected in human [7] and mouse [8, 9] oocytes and in human follicular fluid [7, 10], as well as in granulosa and cumulus cells [7]. Leptin mRNA expression was found in human granulosa and cumulus cells [7] and in pig oocytes at different stages of follicular development and oocyte maturation [11].

The leptin receptor (LEPR) has high sequence homology to the class I cytokine receptor superfamily [12], and alternative 3' terminal mRNA splicing generates several receptor isoforms [12–15]. The long transmembrane isoform containing a 302-residue-long cytoplasmic domain is present in the hypothalamus as well as in peripheral tissues [12, 16]. This isoform is believed to mediate most of the leptin signaling [12, 15, 17] through the signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK) pathways. The other transmembrane receptor isoforms have short cytoplasmic domains and are expressed in many tissues. They have a janus kinase box and are capable of signaling through the MAPK pathway. However, there is evidence that this signaling is weaker than that of the long isoform [16]. Exposure of oocytes to physiological leptin concentrations increases STAT3 [18] and MAPK [11] phosphorylation. Moreover, Lepr/LEPR mRNA is expressed in mouse oocytes [18, 19] and in human granulosa [7, 14], and cumulus cells [7]. Taken together, these findings suggest that leptin modulates function of the oocyte and/or its surrounding cells.

Acquisition of oocyte developmental competence is a limiting step determining the ability of the oocyte to undergo successful fertilization, cleavage. and embryonic development. This final differentiation of the oocyte is orchestrated by a complex network of growth factors and cytokines leading to proper nuclear and cytoplasmic maturation [20]. There is evidence that leptin stimulates oocyte maturation. Leptin concentrations of 10–100 ng/ml increased the proportion of pig oocytes reaching metaphase II stage [11]. In the mouse, culture of follicles in the presence of 10–1000 ng/ml leptin increased oocyte germinal vesicle breakdown [9]. Moreover, leptin administration in rats reduced the incidence of follicular apoptosis [4], implying that leptin can rescue oocytes and follicles from atresia by attenuation of apoptosis. Whereas apoptosis is known to occur in bovine oocytes [21, 22] and preimplantation embryos [23–25], there are few studies on the regulation of apoptosis and gene expression in bovine blastocysts derived from oocytes matured in different controlled systems. Therefore, the objectives of the present study were to determine 1) whether addition of leptin (1, 10 or 100 ng/ml) during maturation of bovine oocytes enhances developmental capacity of oocytes, as reflected by the ability of the oocyte to develop to the blastocyst stage, the number of blastocyst cells, and the incidence of blastocyst apoptosis; and 2) whether changes in these parameters are associated with altered expression of genes relevant for leptin signal transduction or apoptosis-related genes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Recombinant human leptin (purity ≥ 97% as determined by SDS-PAGE; endotoxin contamination ≤ 0.1 ng/µg leptin) was purchased from Sigma Chemical Co. (St. Louis, MO). Bovine pituitary-derived FSH and LH were purchased from Sioux Biochemicals Inc. (Sioux Center, IA). Frozen semen from various bulls was donated by Rinderunion Baden-Württemberg e.V. (Stuttgart, Germany). Tissue culture medium (TCM)-199 with Earle salts was from Biochrom AG (Berlin, Germany). Unless otherwise stated, all reagents used for in vitro production of bovine embryos were purchased from Sigma.

In Situ Cell Death Detection Kit (fluorescein) was obtained from Roche Diagnostics Corporation (Indianapolis, IN). Propidium iodide and polyvinylpyrrolidone (PVP) were purchased from Sigma. Prolong Antifade Kit was obtained from Molecular Probes (Eugene, OR), RQ1 RNA-free DNase was from Promega (Madison, WI), and RNase A was from Qiagen (Hilden, Germany).

Random hexamer primers, TriZol reagent, DNase, RNaseOut, reverse transcriptase enzyme Superscript II, TOPO Cloning Kit, TOPO vector, One Shot chemically competent bacteria, and SOC medium were purchased from Invitrogen (Carlsbad, CA). HotStarTaq polymerase, RNAlater and QIAprep Spin Mini Kit were obtained from Qiagen. Primer Express 2 software, SybrGreen, uracil N-glycosylase, and ABI PRISM 7000 sequence detector system were from Applied Biosystems (Foster City, CA). EcoRI, dNTPs, and XhoI were purchased from Fermentas Life Sciences (Hanover, MD), SeeDNA was from Amersham Biosciences Europe GmbH (Freiburg, Germany), and X-gal was from Carl Roth (Karlsruhe, Germany).

In Vitro Production of Bovine Embryos

Embryos were produced based on procedures described previously [26]. Ovaries were obtained from slaughtered cows and washed several times with PBS (1 mM potassium phosphate, 2 mM potassium chloride, 8 mM sodium phosphate, 0.9 mM calcium chloride, 0.49 mM magnesium chloride and 0.8% sodium chloride) at 25–30°C to remove blood and debris. To obtain COCs, 2–10 mm follicles were aspirated with a 20-gauge needle and a vacuum pressure of approximately 100 mm Hg. COCs that had at least one layer of compact cumulus cells were washed two times in serum-free oocyte maturation medium (TCM-199 with Earle salts containing 22 µg/ml FSH and 8 µg/ml LH) supplemented with 1 mg/ml polyvinylalcohol and used for subsequent steps. Groups of 30 COCs were placed in four-well plates containing 400 µl per well serum-free oocyte maturation medium supplemented with leptin (1, 10 or 100 ng/ml) or 10% (v/v) estrous cow serum (ECS) as a positive control. Oocytes were allowed to mature for 22–24 h at 39°C in an atmosphere of 5% [v/v] CO2 in humidified air. After in vitro maturation, COCs were washed once in IVF-TALP (modified Tyrode stock solution supplemented with 6 mg/ml essentially fatty-acid-free BSA, 0.022 mg/ml pyruvic acid, and 0.01 mg/ml heparin) and transferred to four-well plates containing 400 µl IVF-TALP per well. Spermatozoa from a pool of bulls (two to three bulls) were purified by swim-up procedure in Sperm-TALP (modified Tyrode stock solution supplemented with 6 mg/ml essentially fatty-acid free BSA and 0.11 mg/ ml pyruvic acid) at 39°C in 5% [v/v] CO2 in humidified air for 1 h. Swim-up was followed by centrifugation of spermatozoa from the upper phase for 10 min at 700 x g. The spermatozoa pellet was reconstituted in IVF-TALP and oocytes were fertilized with 25 µl spermatozoa suspension (~1 x 106 spermatozoa/ml). Approximately 18 h after fertilization, presumptive zygotes were denuded of cumulus cells by vortexing in 1 ml synthetic oviduct fluid (SOF) (SOF stock was modified on the day of use by adding 10% [v/v] ECS, 4% [v/v] essential amino acids, and 1% [v/v] nonessential amino acids) for 3 min. Groups of 25–30 presumptive zygotes were placed in 400 µl SOF overlaid with mineral oil and cultured at 39°C in a humidified atmosphere of 5% (v/v) CO2, 5% (v/v) O2, and 90% (v/v) N2.

TUNEL and Propidium Iodide Labeling

The TUNEL procedure was used to detect DNA fragmentation observed in late stages of apoptosis. The enzyme terminal deoxynucleotidyl transferase is a DNA polymerase that catalyzes the transfer of a fluorescein isothiocyanate-conjugated dUTP nucleotide to a free 3' hydroxyl group present in DNA strand breaks. Blastocysts were removed from culture medium (SOF) and washed once in 500 µl PBS (10 mM potassium phosphate, 0.9% [w/v] NaCl, pH 7.4) containing 1 mg/ml polyvinylpyrrolidone (PBS-PVP). Zona pellucida-intact blastocysts were fixed in four-well plates containing 500 µl per well of 4% (w/v) paraformaldehyde in PBS, pH 7.4, for 1 h at room temperature, washed once in PBS-PVP, and stored in 600 µl PBS-PVP at 4°C until TUNEL analysis. On the day of TUNEL staining, blastocysts were permeabilized in four-well plates containing 500 µl permeabilization solution (0.5% [v/v] Triton X-100 containing 0.1% [w/v] sodium citrate) for 1 h at room temperature. Positive and negative controls were incubated in 200 µl RQ1 RNase-free DNase (50 U/ml) at 37°C for 1 h. Blastocysts were washed in PBS-PVP and incubated in a 25 µl drop of TUNEL reaction mixture (containing fluorescein isothiocyanate-conjugated dUTP and the enzyme terminal deoxynucleotidyl transferase), as prepared by the manufacturer, for 1 h at 37°C in the dark. Negative control was incubated in the absence of terminal deoxynucleotidyl transferase. Blastocysts were then incubated in 500 µl RNase A (50 µg/ml) for 1 h at room temperature, followed by 200 µl propidium iodide (0.5 µg/ml) for 30 min at room temperature. Blastocysts were washed three times in PBS-PVP to remove excess propidium iodide, placed on poly-L-lysine coated slides, and mounted with 16 µl mounting medium containing Antifade as recommended by the manufacturer. Each blastocyst was analyzed for total number of nuclei and number of TUNEL-labeled nuclei using a Zeiss Axiovert 200 M fluorescence microscope coupled with the Zeiss filter number 9 (FITC) or 15 (Texas red) as well as the triple band filter 25 (FITC, Texas red and DAPI). Some blastocysts were also examined for photomicroscopy using a Zeiss laser scanning microscope 510 Meta (LSM 510 Meta). For fluorescein, an argon ion laser adjusted to below 560 nm was used, and for propidium iodide, a helium-neon laser adjusted to above 560 nm was used. Digital images were obtained using the LSM 510 Meta system with a 20x objective. Blastocysts derived from oocytes treated with 10 ng/ml leptin had higher cell numbers, and therefore these images were acquired using a 10x objective.

Cloning of Selected Genes

Amplification primers (Table 1) were designed according to sequences found in GenBank and using the software Primer Express 2. Selected primers were submitted to BLAST analysis to check for sequence specificity. Complementary DNA was produced by reverse transcription of bovine ovary and brain total RNA (20 ng/µl). A polymerase chain reaction (PCR) was performed during the primer optimization procedure to determine the presence of specific PCR products and the absence of primer-dimer formation. For each of the selected primers, the PCR reaction included 0.25 µl HotStarTaq (5 IU/µl), 1 µl primer (5 µM), 1 µl cDNA, 2 µl dNTPs (1 mM), 1.25 µl magnesium chloride (25 mM) and 11.5 µl water. The thermal cycler was programmed to run an initial incubation at 95°C for 15 min to activate HotStarTaq. This was followed by 35 PCR cycles of DNA denaturation (95°C for 15 sec), primer annealing (60°C for 30 sec), and elongation (72°C for 30 sec). A sample corresponding to 10% of the PCR products was subjected to 2.5% agarose gel electrophoresis containing ethidium bromide (10 mg/ml). PCR products were visualized by exposure to ultraviolet light.


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  		TABLE 1. Amplification primers

Plasmid standards were established for every gene of interest with the TOPO Cloning Kit as recommended by the manufacturer. The cloned bacteria suspension (One Shot chemically competent bacteria) was spread on an agarose plate containing antibiotic (50 ng/ml ampicillin) and X-gal (50 mg/ml) selection and incubated overnight at 37°C. Selected positive colonies were cultured in Luria-Bertani medium (10 mg/ml tryptone, 5 mg/ ml yeast extract, and 10 mg/ml sodium chloride) containing 50 ng/ml ampicillin overnight at 37°C. The cloned plasmids were isolated using the QIAprep Spin Mini Kit following the manufacturer's instructions. A sample from the isolated plasmids was digested with the restriction endonuclease EcoRI (10 IU/µl) to determine the length of the cloned fragments. Isolated plasmids were linearized using XhoI (10 IU/µl) digestion. Plasmids were subjected to 1% agarose gel electrophoresis as described before. Linearized plasmids with the sequence of interest were diluted to 1 million/µl. To confirm plasmid identity, the PCR products were subjected to sequence analysis (Laboratory for Functional Genome Analysis—LAFUGA, Munich, Germany).

RNA Isolation and Reverse Transcription

Total RNA was isolated from pools of blastocysts (4 blastocysts/tube). TriZol reagent (500 µl) was added to the tube followed by a 15-sec vortexing to homogenize the samples. Samples were centrifuged (10 min, 11 900 x g, 4°C) and the supernatant (450 µl) was mixed with 100 µl chloroform in a new tube, vortexed, and incubated for 10 min at room temperature. Following a second centrifugation step, the top phase (250 µl) was transferred to a new tube and gently mixed with 2 µl of SeeDNA, 0.1 volume of sodium acetate (3 M), and 2 volumes 100% ethanol and incubated for 10 min at room temperature. Samples were centrifuged as described before, and the pellet was washed in 500 µl 75% ethanol and centrifuged again for 5 min. The pellet was dried on air, resuspended in 16.75 µl nuclease-free water, heated for 10 min at 55°C, and chilled on ice. A DNase digestion step was performed using 2 µl DNase digestion buffer and 0.25 µl DNase (1 IU/µl) at 25°C for 15 min. DNase was inactivated with 1 µl EDTA (25 mM) and denaturation at 65°C for 10 min.

For reverse transcription, 2 µl random hexamer primers (3 µg/µl), 2 µl dNTPs (10 mM) and 2.5 µl water were added to the samples, incubated for 5 min at 65°C, and chilled on ice. Samples were subjected to a short centrifugation step and incubated with 8 µl 5x transcription buffer, 4 µl dithiothreitol (0.1 M), and 1 µl RNaseOut (40 IU/µl) for 10 min at 25°C. This was followed by a 2-min incubation step at 42°C and addition of 0.5 µl reverse transcriptase enzyme Superscript II (200 IU/µl). The reverse transcription reaction was carried out for 50 min at 42°C and terminated by incubating the samples for 15 min at 70°C.

Quantitative PCR

Quantitative PCR (qPCR) was performed using the real-time PCR ABI PRISM 7000 sequence detector system and SybrGreen as a double-stranded DNA-specific fluorescent dye. Amplification mixes contained 2 µl cDNA, 12.5 µl SybrGreen PCR Mix, 0.25 µl uracil N-glycosylase (1 IU/ µl), 1.5 µl primer (5 µM), and 7.25 µl water. The ABI PRISM 7000 sequence detector system was programmed to start with uracil N-glycosylase activation for 2 min at 50°C. The program continued with 45 cycles of DNA denaturation (15 sec at 95°C), primer annealing (30 sec at 60°C), and elongation (30 sec at 72°C). Serial dilutions of plasmids were used to establish a standard curve for each gene of interest. Each qPCR was run in triplicate. Transcript copy numbers for LEPR, STAT3, BCL2 associated X-protein (BAX), and baculoviral inhibitor of apoptosis protein repeat-containing 4 (BIRC4, also known as XIAP) were calculated using the standard curve method with determination of PCR amplification efficiency and normalized for histone 2A (H2AFZ) transcript levels [27]. Transcript levels in the control group (0 ng/ml leptin) were set to 1 and data in the treatment groups were calibrated accordingly.

Experiments

Effect of leptin on developmental capacity of bovine oocytes and blastocyst apoptosis COCs were matured in serum-free maturation medium containing 0, 1, 10 or 100 ng/ml recombinant human leptin or maturation medium containing 10% (v/v) ECS as positive control. After 22–24 h maturation, oocytes were subjected to in vitro fertilization and culture. Cleavage rate was assessed at Day 3 and development to the blastocyst stage at Days 7 and 8 postinsemination. This experiment was replicated eight times, using 312–374 oocytes per treatment. Blastocysts from five in vitro production (IVP) replicates (46–72 blastocysts per treatment) were harvested at Day 8 and fixed in 4% paraformaldehyde for TUNEL analysis and determination of total cell number.

Effect of leptin on transcript levels in blastocysts COCs were matured in serum-free maturation medium containing 0, 1, 10 or 100 ng/ml recombinant human leptin or maturation medium containing 10% (v/v) ECS as positive control. After 22–24 h maturation, oocytes were subjected to in vitro fertilization and culture. Expanded and hatched blastocysts were harvested from six IVP replicates (three replicates in May–June 2004 and three replicates in March–April 2005). Expanded blastocysts were harvested at Day 7 and hatched blastocysts at Day 8 postinsemination. For each treatment group, six pools of four expanded and six pools of four hatched blastocysts were analyzed.

Statistical Analysis

Data were analyzed by least-squares analysis of variance. For experiments on the effect of leptin on developmental capacity of bovine oocytes and blastocyst apoptosis, independent variables were treatment and replicate. For experiments on blastocyst gene expression, independent variables were treatment, experimental period, and replicate. Dependent variables were percentage of oocytes cleaved, percentage of blastocyst development, percentage of apoptotic cells, total cell number, mRNA copy number normalized to the housekeeping gene H2AFZ, and the arcsine of the percentage variables. Two analyses were performed: one by the General Linear Models (GLM) procedure of SAS [28], in which replicate (i.e., day of IVP procedure) was considered a fixed variable, and one by the Mixed Models procedure of SAS, where replicate was considered a random variable. Probability values were similar for both methods, and probability values reported herein are derived from the analysis by GLM. Percentage data were analyzed without transformation and after being subjected to the arcsine transformation to correct for problems of nonnormality associated with analysis of percentage data. Probability values were similar for both analyses. Orthogonal contrasts and the PDIFF procedure of SAS were performed when appropriate to determine differences between various levels of a treatment. Data are presented as least squares means ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Leptin on Developmental Capacity of Bovine Oocytes

The purpose of this experiment was to determine whether leptin enhances developmental capacity of bovine oocytes. There was no effect of leptin supplementation during oocyte maturation on cleavage rate after fertilization (81.7 ± 2.1, 85.6 ± 2.0, 85.0 ± 2.0, 81.4 ± 2.0, and 82.0 ± 2.3% cleavage rate for 0, 1, 10 and 100 ng/ml leptin and ECS, respectively). However, an increased (P < 0.05) proportion of oocytes developed to blastocysts in the 10 ng/ ml leptin treatment group at days 7 and 8 and in the 1 ng/ ml leptin treatment group at Day 8 (Fig. 1, A and B). Treatment of oocytes with 100 ng/ml leptin or ECS did not affect their capacity to develop to blastocysts as compared to the serum-free control (Fig. 1).



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  		FIG. 1. Effect of leptin on developmental capacity of bovine oocytes. The proportion of oocytes that developed to the blastocyst stage at Days 7 (A) and 8 (B) postinsemination are shown. Results are presented as least squares means ± SEM of 8 replicates, using 312–374 oocytes per treatment. Significant (P< 0.05) differences from the control group (0 ng/ml leptin) are indicated by an asterisk

Effect of Leptin on Blastocyst Apoptosis

Figure 2, A–D, displays representative confocal images of bovine blastocysts subjected to TUNEL analysis. A blastocyst derived from a control oocyte (0 ng/ml leptin) is shown in Figure 2A. Figure 2, B and C, shows blastocysts derived from oocytes matured in the presence of 1 and 10 ng/ml leptin, respectively. Note that these blastocysts have reduced incidence of TUNEL-positive blastomeres (yellow in color). Indeed, the percentage of blastomeres undergoing apoptosis was reduced in blastocysts originating from oocytes matured in the presence of leptin (1, 10 or 100 ng/ ml, P < 0.001) or ECS (P < 0.001, Fig. 3A). Propidium iodide staining demonstrated that blastocyst cell number was increased by 1 (P < 0.05) and 10 (borderline significance, P = 0.06) ng/ml leptin (Fig. 3B). There was no effect of ECS on blastocyst cell number (Fig. 3B).



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  		FIG. 2. Representative confocal images illustrating the frequency of apoptotic nuclei in bovine blastocysts subjected to TUNEL analysis. Blastocysts were labeled with fluorescein isothiocyanate-conjugated dUTP (green channel) and propidium iodide (red channel). A) A blastocyst derived from control oocytes matured in the absence of leptin (0 ng/ml leptin). Arrows point to TUNEL-positive nuclei. Note the higher frequency of TUNEL-positive blastomeres in the control blastocyst. B and C) Blastocysts derived from oocytes matured in the presence of 1 and 10 ng/ml leptin, respectively. D) Representative picture of a positive control blastocyst pretreated with DNase. Digital images were obtained using the LSM 510 Meta system with a 20x objective. Note that the blastocyst derived from oocytes treated with 10 ng/ml leptin (C) had higher cell numbers, and that therefore the image was acquired using a 10x objective



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  		FIG. 3. Effect of leptin during maturation of bovine oocytes on the proportion of apoptotic cells and total cell number of in vitro derived blastocysts. The proportion of TUNEL-positive blastomeres (A) and blastocyst cell numbers (B) are shown. Results are least squares means ± SEM of five replicates using 46–72 blastocysts per treatment. Significant differences from the control group (0 ng/ml leptin) are indicated by * (P < 0.05) and ** (P < 0.001). Borderline significance is indicated by {dagger} (P = 0.06)

Effect of Leptin on Blastocyst Gene Expression

Messenger RNA levels for H2AFZ, LEPR, STAT3, BAX, and BIRC4 were determined by reverse transcriptase-quantitative PCR (RT-qPCR) to evaluate whether the effect of leptin during oocyte maturation has consequences for the expression of these selected genes at the expanded and hatched blastocyst stage. There was no effect of leptin on expression of the housekeeping gene H2AFZ in expanded and hatched blastocysts. Expanded but not hatched blastocysts originating from ECS-treated oocytes exhibited increased (P < 0.001) H2AFZ mRNA levels. Therefore, the ECS treatment group was not included for the final analysis of transcript levels in expanded blastocysts.

Maturation of bovine oocytes in the presence of physiological doses of leptin increased LEPR mRNA abundance in expanded (1 and 10 ng/ml, P < 0.05) and hatched blastocysts (1 ng/ml, P < 0.05), whereas blastocysts originating from oocytes treated with a higher dose of leptin (100 ng/ ml) tended to have reduced levels of LEPR mRNA (Fig. 4A). ECS had no effect regarding this parameter.



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  		FIG. 4. Effect of leptin during maturation of bovine oocytes on the abundance of LEPR (A), STAT3 (B), BAX (C) and BIRC4 (D) mRNAs in expanded (white bars) and hatched (black bars) blastocysts. Results are least squares means ± SEM of six pools of four expanded and six pools of four hatched blastocysts per treatment group. Significant differences from the control group (0 ng/ml leptin) are indicated by * (P < 0.05) and ** (P < 0.001). Borderline significance is indicated by {dagger} (P = 0.06)

STAT3 mRNA levels were increased in expanded blastocysts derived from the 100 ng/ml leptin treatment group (P < 0.001) and in hatched blastocysts derived from all leptin-treated oocytes (1 and 10 ng/ml, P < 0.001; 100 ng/ ml, P < 0.05). STAT3 transcript levels were increased with borderline significance (P = 0.06; Fig. 4B) in blastocysts originating from ECS-treated oocytes.

BAX transcript levels were reduced in blastocysts derived from 10 ng/ml leptin-treated oocytes (P < 0.001 in expanded and P < 0.05 in hatched blastocysts) and in expanded blastocysts originating from the 100 ng/ml leptin treatment group (P < 0.05, Fig. 4C). There was no effect of 1 ng/ml leptin regarding this parameter at the expanded or hatched blastocyst stage. Moreover, treatment of oocytes with 100 ng/ml leptin or ECS did not affect the level of BAX transcripts in hatched blastocysts (Fig. 4C).

In contrast, transcript levels of the antiapoptotic gene BIRC4 were increased in blastocysts originating from 1 ng/ ml leptin-treated oocytes (P < 0.05 in expanded and P < 0.001 in hatched blastocysts) and the abundance of BIRC4 mRNA was—with borderline significance—also increased in expanded blastocysts from the 100 ng/ml leptin treatment group (P = 0.06, Fig. 4D). Hatched blastocysts derived from other leptin- and ECS-treated oocytes did not exhibit altered BIRC4 transcript levels as compared to the control (Fig. 4D).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrated that leptin at physiological concentrations exerted a beneficial effect during maturation of bovine oocytes. Leptin concentrations (1 and 10 ng/ml) used in the present study represent physiological serum leptin levels found in periparturient [29, 30] and cyclic [31] cows. Leptin improved oocyte developmental competence in a dose-dependent manner. Addition of 1 and 10 ng/ml leptin to serum-free maturation medium did not affect cleavage rate but increased development to the blastocyst stage and blastocyst cell number. It is well known that oocyte developmental potential is a reflection of proper cytoplasmic maturation. Even though most bovine oocytes resume meiosis and progress to metaphase II following in vitro maturation [32], cytoplasmic maturation in vitro is generally compromised, leading to low rates of development. Thus, the positive effect of leptin on the developmental potential of oocytes may be related to their cytoplasmic maturation. Potential modes of action include direct or indirect (cumulus cell-mediated) effects of leptin restructuring oocyte cytoskeleton [33], reprogramming protein synthesis [33], or inhibiting apoptosis [4].

This study shows for the first time that leptin supplementation during bovine oocyte maturation has long-term effects on in vitro-produced blastocysts, i.e., reduction in the percentage of apoptotic cells. Moreover, the reduction in TUNEL-positive cells in blastocysts derived from leptin-treated oocytes was associated with increased BIRC4 and reduced BAX expression. As a member of the inhibitor of apoptosis protein family, BIRC4 acts as an antiapoptotic molecule and blocks apoptosis by inhibiting caspase 3, 7, and 9 [34]. Inhibition of caspases prevents the oligonucleosomal DNA fragmentation typical of apoptosis. In contrast, BAX is a major proapoptotic molecule from the BCL2 protein family [35]. The relative ratio between proapoptotic and antiapoptotic proteins determines the fate of the cell in many cases. Leptin acted in the oocyte and/or cumulus cells causing a long-term shift in the expression of proapoptotic and antiapoptotic genes. It possibly reduced intracellular BAX available to form BAX-BAX homodimers and blocked caspase activity through BIRC4 leading to cell survival.

Apoptosis plays an important role in mammalian development as a quality control mechanism to eliminate cells that are damaged, nonfunctional, abnormal, or misplaced [36, 37]. The occurrence of apoptosis has been demonstrated in many cell types, including bovine oocytes [22], cumulus cells [21, 38], and preimplantation embryos [23, 25, 39–41]. The beneficial effect of leptin during oocyte maturation suggests a role for leptin as a survival factor minimizing cellular damage to oocyte and/or cumulus cells. It is possible that leptin rescued oocytes that would otherwise have generated arrested embryos or embryos with poor developmental potential because of a high incidence of apoptosis. The beneficial effect of leptin during oocyte maturation resulted in blastocysts with reduced apoptosis. Whereas ECS mimicked leptin's effect of minimizing blastocyst apoptosis, it had no effect on the expression of apoptosis-associated genes. The levels of BIRC4 and BAX transcripts in blastocysts were strongly affected by leptin supplementation but not by ECS during oocyte maturation. Thus, it is likely that leptin regulated the apoptotic machinery in a specific manner.

Exposure of oocytes to physiological leptin concentrations resulted in a dose-dependent increase in LEPR and STAT3 mRNA levels in bovine blastocysts. In contrast, LEPR mRNA levels in blastocysts originating from oocytes matured with a high dose of leptin (100 ng/ml) had a tendency to be reduced. Moreover, this high leptin concentration had only a limited effect: it reduced blastocyst apoptosis but had no influence on blastocyst development or blastocyst cell number. A potential reason for the limited effect under this condition might be a dose-dependent downregulation of LEPR mRNA and protein at the oocyte stage. This point needs to be clarified in future studies. Inhibition of LEPR expression by high doses of leptin has previously been observed in other tissues, including hypothalamus [42] and adrenal gland [43]. The fact that STAT3 mRNA levels in blastocysts were also affected by leptin supplementation in the oocyte maturation medium suggests complex and long-lasting regulatory effects of leptin on its signaling machinery.

On the basis of our findings, it is tempting to speculate how leptin might function in the bovine ovary to exert a positive effect on oocytes. Perhaps leptin produced by oocytes and cumulus cells acts in an autocrine/paracrine manner to 1) upregulate LEPR expression; 2) activate STAT3 and MAPK pathways enhancing nuclear and cytoplasmic oocyte maturation; and 3) alter the ratio between antiapoptotic and proapoptotic molecules, reducing cumulus cell and oocyte apoptosis. There is evidence that the long LEPR isoform mediates most of the leptin signaling [12, 15, 17] through the STAT3 and MAPK pathways. Indeed, leptin has been shown to induce STAT3 phosphorylation in mouse oocytes [18] and MAPK phosphorylation in porcine oocytes [11]. In porcine oocytes, leptin improved oocyte maturation through activation of MAPK signaling cascade [11]. It has been suggested that this effect is mediated by the short receptor isoform [11], which has not yet been detected in bovine ovarian tissue [44].

The developmental potential of oocytes matured in vitro is reduced as compared to in vivo oocytes [45]. To characterize the specific requirements for bovine oocyte maturation, it is necessary to establish a serum-free oocyte maturation medium. In the current study, development to the blastocyst stage and blastocyst cell number were not different between oocytes matured in serum-free medium or medium supplemented with 10% serum. Other studies have also reported successful development to the blastocyst stage following serum-free oocyte maturation [46–48]. However, blastocysts derived from oocytes matured under serum-free conditions had increased incidence of apoptosis. This negative effect was prohibited by the addition of leptin to serum-free maturation medium.

In conclusion, leptin treatment during oocyte maturation improved developmental potential, resulting in increased development to the blastocyst stage with reduced numbers of apoptotic cells. Further, increased BIRC4 and reduced BAX mRNA levels as well as effects on transcript levels of LEPR and STAT3 mRNA were detected in blastocysts originating from oocytes of some of the leptin-treatment groups. Thus, physiological doses of leptin during oocyte maturation may have long-term effects on the expression of developmentally important genes in early embryos.


   ACKNOWLEDGMENTS
 
The authors thank Tuna Guengoer for collecting ovaries and blastocysts for RT-qPCR analyses, Myriam Weppert for collecting ovaries, and the Rinderunion Baden-Württemberg e.V. (Stuttgart, Germany) for donating the semen.


   FOOTNOTES
 
1 Supported by the Deutsche Forschungsgemeinschaft (Research Unit "Mechanisms of Embryo-Maternal Communication," FOR 478/1). Back

2 Correspondence: Eckhard Wolf, Department of Molecular Animal Breeding and Biotechnology, Gene Center, Ludwig-Maximilian University, Feodor-Lynen-Strasse 25, 81377 Munich, Germany. FAX: 49 89 2180 76849; ewolf{at}lmb.uni-muenchen.de Back

Received: 17 February 2005.

First decision: 14 March 2005.

Accepted: 14 June 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Caro JF, Sinha MK, Kolaczynski JW, Zhang PL, Considine RV. Leptin: the tale of an obesity gene. Diabetes 1996 45:1455-1462[Medline]
  2. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994 372:425-432[CrossRef][Medline]
  3. Gonzalez RR, Caballero-Campo P, Jasper M, Mercader A, Devoto L, Pellicer A, Simon C. Leptin and leptin receptor are expressed in the human endometrium and endometrial leptin secretion is regulated by the human blastocyst. J Clin Endocrinol Metab 2000 85:4883-4888[Abstract/Free Full Text]
  4. Almog B, Gold R, Tajima K, Dantes A, Salim K, Rubinstein M, Barkan D, Homburg R, Lessing JB, Nevo N, Gertler A, Amsterdam A. Leptin attenuates follicular apoptosis and accelerates the onset of puberty in immature rats. Mol Cell Endocrinol 2001 183:179-191[CrossRef][Medline]
  5. Malik NM, Carter ND, Murray JF, Scaramuzzi RJ, Wilson CA, Stock MJ. Leptin requirement for conception, implantation, and gestation in the mouse. Endocrinology 2001 142:5198-5202[Abstract/Free Full Text]
  6. Coleman DL. Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice. Diabetologia 1978 14:141-148[CrossRef][Medline]
  7. Cioffi JA, Van Blerkom J, Antczak M, Shafer A, Wittmer S, Snodgrass HR. The expression of leptin and its receptors in pre-ovulatory human follicles. Mol Hum Reprod 1997 3:467-472[Abstract/Free Full Text]
  8. Antczak M, Van Blerkom J. Oocyte influences on early development: the regulatory proteins leptin and STAT3 are polarized in mouse and human oocytes and differentially distributed within the cells of the preimplantation stage embryo. Mol Hum Reprod 1997 3:1067-1086[Abstract/Free Full Text]
  9. Ryan NK, Woodhouse CM, Van der Hoek KH, Gilchrist RB, Armstrong DT, Norman RJ. Expression of leptin and its receptor in the murine ovary: possible role in the regulation of oocyte maturation. Biol Reprod 2002 66:1548-1554[Abstract/Free Full Text]
  10. Barroso G, Barrionuevo M, Rao P, Graham L, Danforth D, Huey S, Abuhamad A, Oehninger S. Vascular endothelial growth factor, nitric oxide, and leptin follicular fluid levels correlate negatively with embryo quality in IVF patients. Fertil Steril 1999 72:1024-1026[CrossRef][Medline]
  11. Craig J, Zhu H, Dyce PW, Petrik J, Li J. Leptin enhances oocyte nuclear and cytoplasmic maturation via the mitogen-activated protein kinase pathway. Endocrinology 2004 145:5355-5363[Abstract/Free Full Text]
  12. Tartaglia LA, Dembski M, Weng X, Deng N, Culpepper J, Devos R, Richards GJ, Campfield LA, Clark FT, Deeds J, et al Identification and expression cloning of a leptin receptor, OB-R. Cell 1995 83:1263-1271[CrossRef][Medline]
  13. Chen H, Charlat O, Tartaglia LA, Woolf EA, Weng X, Ellis SJ, Lakey ND, Culpepper J, Moore KJ, Breitbart RE, Duyk GM, Tepper RI, Morgenstern JP. Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/ db mice. Cell 1996 84:491-495[CrossRef][Medline]
  14. Cioffi JA, Shafer AW, Zupancic TJ, Smith-Gbur J, Mikhail A, Platika D, Snodgrass HR. Novel B219/OB receptor isoforms: possible role of leptin in hematopoiesis and reproduction. Nat Med 1996 2:585-589[CrossRef][Medline]
  15. Lee GH, Proenca R, Montez JM, Carroll KM, Darvishzadeh JG, Lee JI, Friedman JM. Abnormal splicing of the leptin receptor in diabetic mice. Nature 1996 379:632-635[CrossRef][Medline]
  16. Bjorbaek C, Uotani S, da Silva B, Flier JS. Divergent signaling capacities of the long and short isoforms of the leptin receptor. J Biol Chem 1997 272:32686-32695[Abstract/Free Full Text]
  17. White DW, Kuropatwinski KK, Devos R, Baumann H, Tartaglia LA. Leptin receptor (OB-R) signaling. Cytoplasmic domain mutational analysis and evidence for receptor homo-oligomerization. J Biol Chem 1997 272:4065-4071[Abstract/Free Full Text]
  18. Matsuoka T, Tahara M, Yokoi T, Masumoto N, Takeda T, Yamaguchi M, Tasaka K, Kurachi H, Murata Y. Tyrosine phosphorylation of STAT3 by leptin through leptin receptor in mouse metaphase 2 stage oocyte. Biochem Biophys Res Commun 1999 256:480-484[CrossRef][Medline]
  19. Kawamura K, Sato N, Fukuda J, Kodama H, Kumagai J, Tanikawa H, Nakamura A, Tanaka T. Leptin promotes the development of mouse preimplantation embryos in vitro. Endocrinology 2002 143:1922-1931[Abstract/Free Full Text]
  20. Greewald GS, Roy SK. Follicular development and its control. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction. New York: Raven Press; 1994:629–724
  21. Kolle S, Stojkovic M, Boie G, Wolf E, Sinowatz F. Growth hormone-related effects on apoptosis, mitosis, and expression of connexin 43 in bovine in vitro maturation cumulus-oocyte complexes. Biol Reprod 2003 68:1584-1589[Abstract/Free Full Text]
  22. Roth Z, Hansen PJ. Involvement of apoptosis in disruption of developmental competence of bovine oocytes by heat shock during maturation. Biol Reprod 2004 71:1898-1906[Abstract/Free Full Text]
  23. Matwee C, Betts DH, King WA. Apoptosis in the early bovine embryo. Zygote 2000 8:57-68[CrossRef][Medline]
  24. Makarevich AV, Markkula M. Apoptosis and cell proliferation potential of bovine embryos stimulated with insulin-like growth factor I during in vitro maturation and culture. Biol Reprod 2002 66:386-392[Abstract/Free Full Text]
  25. Paula-Lopes FF, Hansen PJ. Heat shock-induced apoptosis in preimplantation bovine embryos is a developmentally regulated phenomenon. Biol Reprod 2002 66:1169-1177[Abstract/Free Full Text]
  26. Stojkovic M, Westesen K, Zakhartchenko V, Stojkovic P, Boxhammer K, Wolf E. Coenzyme Q(10) in submicron-sized dispersion improves development, hatching, cell proliferation, and adenosine triphosphate content of in vitro-produced bovine embryos. Biol Reprod 1999 61:541-547[Abstract/Free Full Text]
  27. Robert C, McGraw S, Massicotte L, Pravetoni M, Gandolfi F, Sirard MA. Quantification of housekeeping transcript levels during the development of bovine preimplantation embryos. Biol Reprod 2002 67:1465-1472[Abstract/Free Full Text]
  28. SAS. SAS User's Guide. Cary, NC:. Statistical Analysis System Institute Inc.; 1989
  29. Kadokawa H, Blache D, Yamada Y, Martin GB. Relationships between changes in plasma concentrations of leptin before and after parturition and the timing of first post-partum ovulation in high-producing Holstein dairy cows. Reprod Fertil Dev 2000 12:405-411[CrossRef][Medline]
  30. Soliman M, Ishioka K, Yoshida R, Komabayashi K, Hatai H, Matsui Y, Hirai T, Katagiri S, Takahashi Y, Kawakita Y, Abe H, Kitamura H, Kimura K, Saito M. Serum leptin levels during the periparturient period in cows. J Vet Med Sci 2002 64:1053-1056[CrossRef][Medline]
  31. Garcia MR, Amstalden M, Williams SW, Stanko RL, Morrison CD, Keisler DH, Nizielski SE, Williams GL. Serum leptin and its adipose gene expression during pubertal development, the estrous cycle, and different seasons in cattle. J Anim Sci 2002 80:2158-2167[Abstract/Free Full Text]
  32. Lonergan P, Khatir H, Carolan C, Mermillod P. Bovine blastocyst production in vitro after inhibition of oocyte meiotic resumption for 24 h. J Reprod Fertil 1997 109:355-365
  33. Thibault C, Szollosi D, Gerard M. Mammalian oocyte maturation. Reprod Nutr Dev 1987 27:865-896
  34. Ikeda S, Imai H, Yamada M. Apoptosis in cumulus cells during in vitro maturation of bovine cumulus-enclosed oocytes. Reproduction 2003 125:369-376[Abstract]
  35. Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2002 2:647-656[CrossRef][Medline]
  36. Byrne AT, Southgate J, Brison DR, Leese HJ. Analysis of apoptosis in the preimplantation bovine embryo using TUNEL. J Reprod Fertil 1999 117:97-105
  37. Neuber E, Luetjens CM, Chan AW, Schatten GP. Analysis of DNA fragmentation of in vitro cultured bovine blastocysts using TUNEL. Theriogenology 2002 57:2193-2202[CrossRef][Medline]
  38. Paula-Lopes FF, Hansen PJ. Apoptosis is an adaptive response in bovine preimplantation embryos that facilitates survival after heat shock. Biochem Biophys Res Commun 2002 295:37-42[CrossRef][Medline]
  39. Deveraux QL, Reed JC. IAP family proteins—suppressors of apoptosis. Genes Dev 1999 13:239-252[Free Full Text]
  40. Jacobson MD, Weil M, Raff MC. Programmed cell death in animal development. Cell 1997 88:347-354[CrossRef][Medline]
  41. Meier P, Finch A, Evan G. Apoptosis in development. Nature 2000 407:796-801[CrossRef][Medline]
  42. Martin RL, Perez E, He YJ, Dawson R, Jr. Millard WJ. Leptin resistance is associated with hypothalamic leptin receptor mRNA and protein downregulation. Metabolism 2000 49:1479-1484[CrossRef][Medline]
  43. Tena-Sempere M, Pinilla L, Gonzalez LC, Casanueva FF, Dieguez C, Aguilar E. Homologous and heterologous downregulation of leptin receptor messenger ribonucleic acid in rat adrenal gland. J Endocrinol 2000 167:479-486[Abstract]
  44. Silva LF, VandeHaar MJ, Weber Nielsen MS, Smith GW. Evidence for a local effect of leptin in bovine mammary gland. J Dairy Sci 2002 85:3277-3286[Abstract/Free Full Text]
  45. Lonergan P, Rizos D, Gutierrez-Adan A, Fair T, Boland MP. Oocyte and embryo quality: effect of origin, culture conditions and gene expression patterns. Reprod Domest Anim 2003 38:259-267[CrossRef][Medline]
  46. Saeki K, Hoshi M, Leibfried-Rutledge ML, First NL. In vitro fertilization and development of bovine oocytes matured in serum-free medium. Biol Reprod 1991 44:256-260[Abstract]
  47. Gardner DK. Mammalian embryo culture in the absence of serum or somatic cell support. Cell Biol Int 1994 18:1163-1179[CrossRef][Medline]
  48. Eckert J, Niemann H. In vitro maturation, fertilization and culture to blastocyst of bovine oocytes in protein-free media. Theriogenology 1995 43:1211-1225



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