Mitochondrial biology in reproduction

Abstract

Mitochondrial biology plays an important role in the reproductive process, with influence on germ cell development and quality as well as embryonic development and reproductive success. This review outlines the role of mitochondrial genetics and function in reproductive biology, including a discussion of general mitochondrial function, genetics and germline transmission. Also highlighted are the mitochondrial morphologic changes that occur during oogenesis and the role these changes play in the mitochondrial bottleneck that influences the distribution of deleterious mitochondrial genomes to offspring. The review covers the influence of mitochondria in embryonic stem cell and induced pluripotent stem cell biology and development. Lastly, the role of mitochondrial biology in assisted reproductive techniques is discussed.

Introduction

Life is a constant battle against entropy. In order to maintain the organization and biological processes inherent to life, energy is required. One of the most critical processes that any organism undertakes is the process of reproduction. Reproduction allows a species to maintain existence and allows evolution to shape life over generations. Of more immediate concern perhaps, reproduction allows individuals to build families, and allows the production of food through agriculture. Given the importance of reproduction, the specifics of mitochondrial energy production during reproduction represent an important area of study. Scientists are gaining a better appreciation for the importance of mitochondrial biology in reproduction and this is highlighted by the increase in publications investigating their interconnectivity (Fig. 1). Clearly, there are significant mitochondrial inter‐relationships and functional considerations when investigating reproductive phenomena. The specifics of the maternal inheritance of mitochondrial DNA (mtDNA) and mitochondrial function are of critical importance to the study of reproductive systems.

Figure 1.

Number of publications on mitochondrial biology in reproduction from 1990 to 2010. A PubMed search for the terms ‘mitochondr*’ and ‘reproduction’ for each year since 1990 illustrates a dramatic increase in publication/interest

Mitochondria are subcellular organelles, believed to have originated when our proto‐eukaryotic ancestors either trapped or formed a symbiotic relationship with another organism that eventually became mitochondria [1]. Following integration of the two organisms, mitochondrial biology has become an integral aspect of the study of life.

Mitochondrial bioenergetics

Mitochondria produce energy in the form of adenosine triphosphate (ATP) for the cell through the process of oxidative phosphorylation. The process of energy production begins with the breakdown of glucose through glycolysis into two molecules of pyruvate. Pyruvate is converted into acetylcoenzyme A (acetyl‐CoA), producing nicotinamide adenine dinucleotide (NADH). The acetyl‐CoA is combined with a four‐carbon molecule, oxaloacetic acid, as it is shuttled into the tricarboxylic acid (TCA) cycle, also known as the Krebs cycle. This six‐carbon molecule, citric acid, is broken down throughout the TCA cycle into oxaloacetic acid, producing NADH, flavin adenine dinucleotide (FADH₂) and carbon dioxide waste. Complexes I and II of the electron transport chain (ETC) utilize NADH and FADH₂, respectively, to pass electrons to complex III and shuttle protons from the mitochondrial matrix across the inner mitochondrial membrane, creating a hydrogen ion gradient. Complexes III and IV of the ETC utilize the flow of electrons from complexes I and II to shuttle more protons out of the mitochondrial matrix. Complex IV of the ETC combines the transferred electron with protons and oxygen within the mitochondrial matrix to produce water byproduct, preventing the electrons from forming reactive radicals, which can be harmful when produced in excess. Complex V of the ETC utilizes the proton gradient set up by complexes I through IV, allowing protons to flow down the electrochemical gradient, causing rotation of the gamma subunit. The rotational motion produces conformational changes in the alpha and beta subunits of complex V, which phosphorylate adenosine diphosphate into ATP. The ATP is transferred out of the mitochondria by the adenine nucleotide translocase complex [1].

Mitochondrial genetics

Mitochondria are unique among mammalian organelles in that they contain their own genetic material that is transcribed and translated within the mitochondria. In mammals, the mtDNA is a circular DNA molecule roughly 16,000 bases in length encoding 22 tRNAs, 13 proteins and 2 rRNAs. The transcription machinery is nuclear encoded and the proteins are imported into the mitochondria for mtDNA transcription. The control region of the mtDNA contains the displacement loop (D‐loop) and the three mitochondrial promoters, light strand promoter, heavy strand promoter 1 and heavy strand promoter 2. Transcription proceeds bi‐directionally along both heavy and light strands to produce polycistronic RNAs, which are then processed into the individual mRNAs, tRNAs and rRNAs which are required within the mitochondria for translation. Mitochondrial translation occurs within the mitochondrial matrix and the proteins produced are then assembled into ETC complexes [2, 3].

Maternal inheritance

Mitochondrial DNA is inherited from mother to offspring, which enables maternal lineage tracking. A mammalian oocyte contains from 150,000 to over one million copies of the mtDNA in a species‐dependent manner, with a large variation observed both between and within individual reports [4, 5, 6, 7, 8, 9]. The oocyte passes these copies of the mtDNA to the developing embryo after fertilization, while mitochondria introduced by the sperm are degraded, preventing paternal mtDNA inheritance. In mammals, the elimination of paternal mtDNA from the oocyte occurs by a proteolytic degradation pathway targeting the ubiquitinated paternal mitochondria [10, 11, 12].

Mitochondria morphology and function in reproductive cells

Mitochondrial function in oocytes and developing embryos correlates with growth rates and successful fetal development. Several aspects of mitochondrial biology influence oocyte quality and the likelihood of successful embryo development, including gross mitochondrial morphology, mitochondrial mtDNA quantities and mitochondrial distribution [7, 9, 13, 14].

Mitochondrial morphology undergoes drastic changes throughout the process of oogenesis, fertilization and embryonic development. Specific changes to mitochondrial morphology have profound implications for energy production during reproduction. It is well accepted that changes to mitochondrial morphology affect energy production, with elongated mitochondria producing more ATP than fragmented mitochondrial populations [15]. In differentiated cells, mitochondria form an extensive interconnected network of tubular organelles surrounding the nucleus, with smaller mitochondria distributed throughout the cytoplasm [15]. During oogenesis, mitochondria are initially present in low numbers and exhibit spherical morphology with reduced cristae. Mitochondria begin to replicate in the primordial follicle, and maintain the morphology seen in oogonium. Mitochondrial numbers begin to increase greatly in primary oocytes and replication continues until the oocyte is mature, and then mitochondrial replication stops until post‐implantation [4, 14, 16].

Spherical mitochondrial morphology suggests reduced mitochondrial function, and the literature supports this concept through studies showing that granulosa cells support developing oocytes metabolically. Granulosa cells connect to the developing oocyte through gap junctions and supply the developing oocyte with ATP and pyruvate [17, 18]. In a striking demonstration, research showed that granulosa cell metabolic support is adequate to allow oocytes with severely impaired pyruvate dehydrogenase activity, a key enzyme for energy production, to develop into mature oocytes, ovulate and be fertilized, whereupon they were incapable of embryonic development, presumably due to metabolic insufficiency [18]. Successful oogenesis and ovulation without pyruvate dehydrogenase activity demonstrates the high level of metabolic support provided by granulosa cells. Support of the oocyte by granulosa cells may allow oocytes to be metabolically ‘turned off’ preventing excess reactive oxygen species from damaging mitochondrial and genomic DNA. Effectively, this mechanism could represent another way to minimize mutation transmission between generations. The increased mitochondrial number in mature oocytes would then allow oocytes to meet the energetic demands of fertilization and development without the metabolic support of the granulosa cells.

Another aspect of mitochondrial biology affected by mitochondrial morphology in oocytes is the equal inheritance of mitochondrial number during early cellular division of the embryo [4]. The even distribution of mitochondria of reduced size facilitates even distribution during mitosis. If mitochondria maintained their highly elongated and tubular network morphology characteristic of differentiated cells, uneven distribution of mitochondria to daughter cells during embryo development might cause severe developmental dysfunction [19].

Mitochondrial function is also of critical importance to sperm quality. Alterations or deficiencies in mitochondrial function have been associated with sperm number, vitality and motility [20, 21, 22]. Reports on the metabolic characteristics of sperm in the literature offer little consensus on the role played by mitochondrial respiration despite the association between mitochondrial dysfunction and male infertility. Reports show that activated boar spermatozoa utilize glycolysis almost exclusively and that very little pyruvate produced in spermatozoa enters the Krebs cycle [23]. It was noted that glycogen synthesis was absent in boar spermatozoa, whereas dog spermatozoa maintained active glycogen synthesis. The authors propose that sperm metabolism varies dramatically between species, potentially as an adaption to the environment of the female genital tract, allowing for differences in sperm motility and nutrient requirements. Research by others has demonstrated the importance of glycolysis for sperm motility. A sperm‐specific knockout of glyceraldehyde‐3‐phosphate dehydrogenase, a key enzyme in glycolysis, leads to severe decreases in sperm motility while a sperm‐specific knockout of cytochrome c, a key enzyme in oxidative phosphorylation, results in only moderately reduced sperm motility [24].

Given evidence for the minimal reliance of oxidative phosphorylation in activated spermatozoa, it is paradoxical that various models and clinical studies have shown a correlation between mtDNA mutations, mitochondrial function and sperm quality and fertility [20, 21, 22, 25]. In many cases, impaired mitochondrial function leads to defective spermatogenesis, leading to reduced spermatozoa quantity and malformed spermatozoa. Therefore, oxidative phosphorylation appears to be critical for spermatogenesis, while reduced oxidative phosphorylation in spermatozoa has little effect on motility and fertility rates. Impaired sperm motility seen in models of mitochondrial dysfunction is likely due to malformed spermatozoa produced during dysfunctional spermatogenesis. Research supports this concept through studies demonstrating a link between mitochondrial dysfunction, low sperm motility and malformed spermatozoon [22, 25]. Therefore, the correlation between reduced sperm motility and impaired mitochondrial function or mtDNA mutations likely results from defects during spermatogenesis rather than energetic deficiencies in activated spermatozoa.

The mitochondrial bottleneck

mtDNA mutations can lead to severe metabolic disorders [26, 27]. An important concept when studying diseases associated with mtDNA mutations is heteroplasmy. Heteroplasmy is a measure of the number of mutated mtDNA genomes compared to the total number of mtDNA molecules, generally shown as a percentage. Low heteroplasmy levels of mutations, in the range of 0–50% heteroplasmy, generally do not cause disease, while higher heteroplasmy levels lead to increasingly severe symptoms. Heteroplasmy can vary widely within cells, tissues and organs. The mtDNA heteroplasmy of an individual is determined through a biological phenomenon known as the mitochondrial ‘bottleneck’ effect, which can dramatically change offspring heteroplasmy compared to maternal heteroplasmy [1, 26].

There are two primary views on the mechanisms of the mitochondrial bottleneck effect (Fig. 2). One view is that there is a dramatic reduction in mtDNA molecules during primordial germ cell or oocyte development, allowing a ‘sampling’ effect to determine offspring heteroplasmy. In the mature oocyte and pre‐implantation embryo, total mtDNA genomes do not increase, and may decrease in some species [6, 14, 19]. This means that as the fertilized oocyte begins to divide, the total mtDNA number per cell halves with each subsequent division until mtDNA replication resumes in the post‐implantation blastocyst. Thus, a severely reduced number of mtDNA molecules are present in the primordial germ cells, causing the mitochondrial bottleneck. Measurements of mtDNA copy numbers in isolated primordial germ cells have revealed that as few as ~250 mtDNA molecules may be present in 7.5 days post coitum mouse embryo primordial germ cells [28, 29]; however, there is disagreement on this number and whether there is significant variance in heteroplasmy levels in primordial germ cells of this stage [4, 30].

Figure 2.

Representation of two theories of the mitochondrial bottleneck in oogenesis. Estimated average mtDNA copy number per cell is presented, with variance in heteroplasmy presented adjacent to each theory. Theory 1 holds that the mitochondrial bottleneck occurs during mtDNA copy number reduction in primordial germ cells. Theory 2 proposes that the mitochondrial bottleneck occurs later in development, during oogenesis due to selective replication of a subset of mtDNAs within a cell

An opposing view is that during primordial germ cell or oocyte development, a small portion of the mtDNA within the cell replicates, which causes dramatic changes to heteroplasmy within the cell [4, 30]. Evidence for this perspective comes from work analyzing mtDNA from primordial germ cells. Measurements confirmed a reduction in mtDNA number in primordial stem cells. Yet, quantification of single‐cell heteroplasmy in primordial stem cells and daughter cells did not demonstrate the anticipated variance in heteroplasmy between primordial germ cells. Increased variation began only in developing oocytes, indicating that reduced primordial germ cell mtDNA copy number does not affect heteroplasmy [4, 31]. Rather, heteroplasmy shifts occur later during germ cell development. Challenges to data interpretations supporting this mitochondrial bottleneck theory center on data analysis and statistical methodologies utilized to calculate variance in heteroplasmy [31, 32].

The cause of the mitochondrial bottleneck effect remains an open question. Future research precisely quantifying mtDNA heteroplasmy levels and mtDNA copy numbers in individual cells at various stages of development of the female germ cell will be critical to determine the biological mechanisms behind the mitochondrial bottleneck. Additionally, innovative use of animal models harboring heteroplasmic mtDNA will prove invaluable to uncovering mechanisms of mtDNA heteroplasmy changes occurring through generations [33].

Mitochondrial function and iPS cell biology

In reproductive studies or reproductive technologies, the use of embryonic stem (ES) cells is common [34]; however, moral and legal concerns surround the use of human ES cells. Recent developments in the production of induced pluripotent stem (iPS) cells derived from adult cells has provided a way to avoid many problems associated with using human ES cells [35]. However, questions remain over differences between ES and iPS cells and answers are critical to the development of any model systems or therapeutics utilizing iPS cells.

ES cells derived from the inner cell mass of a blastocyst have strikingly different mitochondrial morphology and function compared to somatic cells. The importance of mitochondrial biology in ES cells was recently highlighted by the observation that inhibition of oxidative phosphorylation maintains ES cells in an undifferentiated state [36]. Several studies have begun to investigate mitochondrial function and morphology in iPS cells. Improper mitochondrial function or morphology in iPS cells could negatively affect normal growth or differentiation. Several studies characterizing iPS cells demonstrated mtDNA quantity and mitochondrial morphology similar to that seen in ES cells with reduced mitochondrial size and reduced mitochondrial cristae [37, 38]. Furthermore, biochemical and genetic studies have shown similarities between ES and iPS cell mitochondrial respiration, oxidative species scavenging capabilities and the expression patterns of many genes with a role in mitochondrial biology [37, 38]. Differences evident in the expression of genes involved in mitochondrial function, such as uncoupling proteins 2 and 4, present the question of the functional consequence of such differences in iPS cells [37, 38, 39].

If metabolic reprogramming in iPS cells is incomplete, or if the mtDNA of iPS cells harbors more polymorphisms than ES cells, metabolic dysfunction may result. Additionally, the mitochondrial bottleneck that can eliminate severe mtDNA mutations may not occur when producing animals using iPS cell‐based technologies. This could result in both deleterious as well as beneficial implications. The mitochondrial bottleneck is a selection mechanism that can radically change heteroplasmy between generations. This shift has the benefit of potentially eliminating or severely decreasing mutant mtDNAs in offspring, and preventing the transmission of highly polymorphic mtDNA populations across generations. Without the bottleneck phenomenon, individual offspring would instead harbor heterogeneous mtDNA populations. This would ensure, minimally, that individual offspring would possess a low abundance of deleterious mtDNA mutations. However, by eliminating the mitochondrial bottleneck, production of animals using iPS cells creates the potential for highly polymorphic and heteroplasmic mtDNA populations in successive generations; this could readily lead to hallmarks of mitochondrial dysfunction and disease ranging from developmental to respiratory deficits.

While there is obviously trepidation in purposefully modifying mitochondrial genetics in developing oocytes, there is a potential benefit in circumventing the mitochondrial bottleneck in females harboring heteroplasmic mtDNA mutations. Conceivably, offspring produced using maternally‐derived iPS cells would have a lower probability of producing offspring with radically different heteroplasmic states. Such outcomes would allow production of numerous healthy offspring from a female that otherwise may produce offspring with metabolic deficiencies.

However, taken collectively for a host of therapies exploiting iPS cell technologies, there are potential drawbacks related to mitochondrial genetics that must be further characterized.

Mitochondria and assisted reproductive technology

The role of mitochondria in assisted reproductive technology is an active field of study. Manipulation of oocytes or embryos to enhance developmental potential involves alteration or introduction of mitochondria in many cases [6, 40, 41, 42].

During cytoplasmic transfer, injection of cytoplasm from healthy oocytes or granulosa cells into an oocyte aims to supplement the oocyte with healthy mitochondria, allowing normal embryonic development. Cytoplasmic transfer resulted in the birth of several children from mothers experiencing reproductive difficulties [43, 44]. This procedure, however, creates heteroplasmic offspring harboring both endogenous and introduced mtDNA. While negative effects may or may not be anticipated from heteroplasmy induced through these methods, further studies are required to test the effects experimentally [45, 46, 47, 48]. Other assisted reproductive technologies also have the potential to create heteroplasmic offspring, including germinal vesicle transfer, pronuclear transfer and somatic cell nuclear transfer [6, 41]. The creation of heteroplasmic animals and transfer of mitochondria between species also provides the potential to study metabolic, developmental and mitochondrial diseases (Fig. 3) [27, 28, 40, 42, 47, 48, 49, 50, 51, 52, 53, 54]. Heteroplasmic animals were used effectively to study the mitochondrial bottleneck, distribution of mitochondria during embryonic development, elimination of deleterious mtDNA mutations, and ETC dysfunction caused by mismatch between nuclear and mtDNA‐encoded subunits of the ETC [4, 50, 51, 55]. Accordingly, future studies using heteroplasmic animals could also allow production of agricultural animals with altered metabolic traits.

Figure 3.

Mouse models of mitochondrial dysfunction created through interspecies mitochondrial transfer. a Chimeric animals are produced by injection of ES cells into blastocysts. Note the chimeric mouse on the right with the presence of both light (agouti) and dark (black) fur. Coat color chimerism is indicative of tissues derived both from 129S6 cells (generating the agouti coat color) and C57BL/6 cells (and resultant black fur). The mouse on the left is a control C57BL/6 mouse. b Chimeric females give birth to homoplasmic offspring derived either from C57BL/6 cells (black pups) or xenomitochondrial 129S6 cells (agouti pup on right). A very low percentage chimeric dam is pictured with her offspring (adapted from [56]). c Normal (black) and xenomitochondrial (agouti) littermate pups derived from chimeric females. d Xenomitochondrial females mated to C57BL/6 males produce homoplasmic offspring with both black and agouti coat colors

Conclusion

Studies investigating the role of mitochondria in reproductive biology enhance our understanding of the mechanisms by which organisms reproduce. Additionally, such studies reveal the specifics of how alterations in energy production mechanisms can influence the biology of developing organisms. They also demonstrate how modulation of energy production mechanisms can foster improvement of many current technologies employed for enhancing reproductive success. Future studies will undoubtedly continue to reveal the importance of mitochondrial biology in reproductive processes. Ultimately, they will allow researchers the ability to develop and advance assisted reproductive technologies to further the goals of both human and animal reproduction.

Acknowledgments

We gratefully acknowledge M.H. Irwin, D.A. Dunn, K. Parameshwaran, F.F. Bartol, K. Steliou and I.A. Trounce. Mitochondrial studies were supported by the US National Institutes of Health, US National Science Foundation, the MitoCure Foundation, the Alabama Agricultural Experiment Station and Auburn University.