Dr. Bo Xiong (far right) and team members (from left to right) Ms. Yu Zhang, Mr. Zhaokang Cui and Ms. Wenjun Zeng in front of their ZEISS LSM 900 confocal microscope, College of Animal Science and Technology, Nanjing Agricultural University, China

Fading Fertility: Using Confocal Microscopy to Understand Oocyte Quality

Approximately every month, primary oocytes in fertile women develop into a few ova (egg cells); if one is fertilized with sperm, it can create a new human. The production of these cells actually starts during a woman’s embryonic development and the primary oocytes exist in a partially developed state until just prior to ovulation, when they complete meiosis to form eggs that are released from the ovary. Some primary oocytes exist in this partially developed state – with their DNA replicated into sister chromatids and paired up with homologous chromosomes, waiting for separation into mature eggs – for upwards of forty years.

Dr. Bo Xiong is a reproductive biologist at the College of Animal Science and Technology, Nanjing Agricultural University, China. He has been working in this field for over 20 years.

He recently published a paper which used confocal microscopy to uncover new roles of proteins involved in producing fertilization-competent eggs in mice and pigs. We spoke to him about his research.

Tell us about the goals of your lab.

The research area of my lab is focused on oocyte quality control. As we know, the high quality of oocytes, a precondition for successful fertilization and subsequent embryonic development, is the material basis for onset of life. In most mammals, female reproductive aging is primarily featured by a prominent decline in the quantity and quality of oocytes. It has been reported that the fecundity of women begins to drop in their early 30s and falls more rapidly after the age of 35, accompanied by a dramatically increased incidence of infertility, miscarriage, embryo lethality and congenital birth defects. Thus, the low quality of oocytes is a common and insurmountable problem for women with advanced maternal age.

In spite of the significance of the problem, strategies to sustain oocyte quality with age have been poorly explored. Our work aims to identify the molecular biomarkers for oocyte quality and develop effective approaches and strategies to protect the oocytes from deterioration induced by maternal aging, contributing to improved fertility and enhanced efficiency of assisted reproductive technology.

Spindle assembly and chromosome alignment in mouse oocyte. Mouse oocyte at metaphase I stage was immunostained with α-tubulin-FITC antibody to show the spindle morphology (green),  with CREST to display the kinetochores (red), and counterstained with Hoechest to visualize the chromosome (blue). Image acquired with ZEISS LSM 900. Courtesy of B. Xiong, College of Animal Science and Technology, Nanjing Agricultural University, China.

Spindle assembly and chromosome alignment in mouse oocyte. Mouse oocyte at metaphase I stage was immunostained with α-tubulin-FITC antibody to show the spindle morphology (green), with CREST to display the kinetochores (red), and counterstained with Hoechest to visualize the chromosome (blue). The proper organization of spindle/chromosome structure drives the oocyte nuclear maturation. Image acquired with the ZEISS LSM 900 confocal microscope.

What are your recent discoveries?

Cohesin is a well-studied protein complex important for sister chromatid cohesion, chromosome segregation as well as many other DNA-related activities. Sororin is an accessory protein to cohesin and acts as a stabilizer for cohesin during cell division. In our recent article, published in Science Advances, we found that sororin also has a non-canonical role during oocyte meiotic progression into mature eggs.

Distribution of cortical granules (CGs) in mouse oocyte. Mouse oocyte at GV (germinal vesicle)  stage was stained with LCA (lens culinaris agglutinin)-FITC to show the CGs (cortical granules, green),  and counterstained with PI (propidium iodide) to visualize the nucleus (red). Image acquired with ZEISS LSM 900. Courtesy of B. Xiong, College of Animal Science and Technology, Nanjing Agricultural University, China.

Distribution of cortical granules (CGs) in mouse oocyte. Mouse oocyte at GV (germinal vesicle) stage was stained with LCA (lens culinaris agglutinin)-FITC to show the CGs (cortical granules, green), and counterstained with PI (propidium iodide) to visualize the nucleus (red). The normal distribution of CGs under the oocyte subcortex is one of the key indicators for oocyte cytoplasmic maturation. Image acquired with the ZEISS LSM 900 confocal microscope.

We provide a body of evidence documenting that sororin acts as a regulator of mammalian ooctyes as they begin cell division and creation of the meiotic spindle that will separate homologous chromosomes. These meiotic functions of sororin are conserved among at least two species, mouse and pig. Our findings not only discover a novel function and an unknown downstream effector of sororin during female egg development, but also extend our understanding of the molecular basis underlying the etiology of oocyte maturational arrest in humans.

How did confocal microscopy assist your research?

For our studies of sororin and its involvement with mammalian oocytes, we must be able to clearly visualize the dynamics of organelles, such as the microtubule organization of the meiotic spindle, chromosome alignment, kinetochore-microtubule attachment, actin polymerization, mitochondrial distribution and cortical granule translocation. The ZEISS LSM 900 confocal microscope allows us to elegantly investigate protein localization, co-localization and fluorescent signal quantification. In particular, 3D imaging was critical when counting the number of sperm binding to the zona pellucida surrounding the whole egg, a critical indicator for evaluating the fertilization ability.

The binding of mouse sperm to the mouse egg. Mouse sperm binding to the zona pellucida of mouse egg were stained with Hoechst to show the sperm head (blue). Image acquired with ZEISS LSM 900. Courtesy of B. Xiong, College of Animal Science and Technology, Nanjing Agricultural University, China.

The binding of mouse sperm to the mouse egg. Mouse sperm binding to the zona pellucida of mouse egg were stained with Hoechst to show the sperm head (blue). The number of sperm binding to the zona pellucida surrounding the whole egg is a critical indicator for evaluating fertilization ability. Image acquired with the ZEISS LSM 900 confocal microscope.

Where do you see your research going next?

Since the first ever in vitro fertilization (IVF) birth occurred in 1978, which led Robert Edwards receiving a Nobel Prize in 2010, this assisted reproductive technology (ART) has produced more than 8 million babies up to now. In spite of its great clinical success, IVF is mainly associated with abnormal polyspermy, due to the dispermic oocyte penetration resulting in tripronuclear (3PN) oocytes. The prevention and elimination of polyspermic oocytes are critical as it has known that significantly higher spontaneous abortions and embryonic death are expected from the transfer of embryos derived from triploid zygotes. Thus, how only one sperm fertilizes each egg becomes one of the central questions about mammalian fertilization.

After a century investigations in the field, we now understand that to prevent polyspermy, the egg develops the plasma membrane and zona pellucida blocks to post-fertilization fusion, penetration and binding of additional sperm. It has been identified that Juno is implicated in the plasma membrane block and ovastacin is responsible for zona block to sperm binding. Nevertheless, the molecular basis and underlying mechanism for the post-fertilization zona pellucida block to sperm penetration has not been uncovered. The next goal of our lab is dedicated to address this scientific question.

Chromosome spreading was performed to assess the euploidy of mouse oocyte at metaphase II stage. Matured oocyte was immunostained with CREST to display the kinetochores and counterstained with Hoechst to show the chromosomes. Image acquired with ZEISS LSM 900. Courtesy of B. Xiong, College of Animal Science and Technology, Nanjing Agricultural University, China.

Chromosome spreading was performed to assess the euploidy of mouse oocyte at metaphase II stage. Matured oocyte was immunostained with CREST to display the kinetochores and counterstained with Hoechst to show the chromosomes. The correct number of chromosomes is required for euploidy maintenance and genome stability in matured oocytes. Image acquired with the ZEISS LSM 900 confocal microscope.

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