Determinants of thermal homeostasis in the preimplantation embryo: a role for the embryo's central heating system?

A number of factors may impinge on thermal homeostasis in the early embryo. The most obvious is the ambient temperature in which development occurs. Physiologically, the temperature in the lumen of the female tract is typically lower than the core body temperature, yet rises at ovulation in the human, while in an IVF setting, embryos are usually maintained at core body temperature. However, internal cellular developmental processes may modulate thermal control within the embryo itself, especially those occurring in the mitochondria which generate intracellular heat through proton leak and provide the embryo with its own 'central heating system'. Moreover, mitochondrial movements may serve to buffer high local intracellular temperatures. It is also notable that the preimplantation stages of development would generate proportionally little heat within their mitochondria until the blastocyst stage as mitochondrial metabolism is comparatively low during the cleavage stages. Despite these data, the specific notion of thermal control of preimplantation development has received remarkably scant consideration. This opinion paper illustrates the lack of reliable quantitative data on these markers and identifies a major research agenda which needs to be addressed with urgency in view of laboratory conditions in which embryos are maintained as well as climate change-derived heat stress which has a negative effect on numerous clinical markers of early human embryo development.

Glucose concentration during equine in vitro maturation alters mitochondrial function.

The use of in vitro embryo production in the horse is increasing in clinical and research settings; however, protocols are yet to be optimised. Notably, the two most commonly used base media for in vitro maturation (IVM) supply glucose at markedly different concentrations: physiological (5.6 mM, M199) or supraphysiological (17 mM, DMEM/F-12). Exposure to high glucose has detrimental effects on oocytes and early embryos in many mammalian species, but the impact has not yet been examined in the horse. To address this, we compared the energy metabolism of equine COCs matured in M199-based maturation medium containing either 5.6 or 17 mM glucose, as well as expression of key genes in oocytes and cumulus cells. Oocytes were fertilised by ICSI and cultured. Analysis of spent medium revealed that COC glucose consumption and production of lactate and pyruvate were similar between treatments. However, the glycolytic index was decreased at 17 mM and analysis of mitochondrial function of COCs revealed that IVM in 17 mM glucose was associated with decreased ATP-coupled respiration and increased non-mitochondrial respiration compared to that for 5.6 mM glucose. We also found that the metabolic enzyme lactate dehydrogenase-A (LDHA) was downregulated in cumulus cells of oocytes that completed IVM in 17 mM glucose. There was no difference in maturation or blastocyst rates. These data indicate that COC mitochondrial function and gene expression are altered by high glucose concentration during IVM. Further work is needed to determine if these changes are associated with developmental changes in the resulting offspring.

Modelling oviduct fluid formation in vitro.

Oviduct fluid is the microenvironment that supports early reproductive processes including fertilisation, embryo cleavage, and genome activation. However, the composition and regulation of this critical environment remains rather poorly defined. This study uses an in vitro preparation of the bovine oviduct epithelium, to investigate the formation and composition of in vitro derived oviduct fluid (ivDOF) within a controlled environment. We confirm the presence of oviduct specific glycoprotein 1 in ivDOF and show that the amino acid and carbohydrate content resembles that of previously reported in vivo data. In parallel, using a different culture system, a panel of oviduct epithelial solute carrier genes, and the corresponding flux of amino acids within ivDOF in response to steroid hormones were investigated. We next incorporated fibroblasts directly beneath the epithelium. This dual culture arrangement represents more faithfully the in vivo environment and impacts on ivDOF composition. Lastly, physiological and pathophysiological endocrine states were modelled and their impact on the in vitro oviduct preparation evaluated. These experiments help clarify the dynamic function of the oviduct in vitro and suggest a number of future research avenues, such as investigating epithelial-fibroblast interactions, probing the molecular aetiologies of subfertility, and optimising embryo culture media.

Biological optimization, the Goldilocks principle, and how much is lagom in the preimplantation embryo.

The quiet embryo hypothesis postulates that early embryo viability is associated with a relatively low metabolism (Leese, 2002 BioEssays 24: 845-849). This proposal is re-visited here using retrospective and prospective data on the metabolic activity and kinetics of preimplantation development alongside the concept that an optimal range of such indices and of energetic efficiency influences embryogenesis. It is concluded that these considerations may be rationalized by proposing the existence of a "Goldilocks zone," or as it is known in Sweden, of lagom-meaning "just the right amount"-within which embryos with maximum developmental potential can be categorized. Mol. Reprod. Dev. 83: 748-754, 2016 © 2016 Wiley Periodicals, Inc.

Human embryos from overweight and obese women display phenotypic and metabolic abnormalities.

STUDY QUESTION: Is the developmental timing and metabolic regulation disrupted in embryos from overweight or obese women? SUMMARY ANSWER: Oocytes from overweight or obese women are smaller than those from women of healthy weight, yet post-fertilization they reach the morula stage faster and, as blastocysts, show reduced glucose consumption and elevated endogenous triglyceride levels. WHAT IS KNOWN ALREADY: Female overweight and obesity is associated with infertility. Moreover, being overweight or obese around conception may have significant consequences for the unborn child, since there are widely acknowledged links between events occurring during early development and the incidence of a number of adult disorders. STUDY DESIGN, SIZE, DURATION: We have performed a retrospective, observational analysis of oocyte size and the subsequent developmental kinetics of 218 oocytes from 29 consecutive women attending for ICSI treatment and have related time to reach key developmental stages to maternal bodyweight. In addition, we have measured non-invasively the metabolic activity of 150 IVF/ICSI embryos from a further 29 consecutive women who donated their surplus embryos to research, and have related the data retrospectively to their body mass index (BMI). PARTICIPANTS/MATERIALS, SETTING, METHODS: In a clinical IVF setting, we compared oocyte morphology and developmental kinetics of supernumerary embryos collected from overweight and obese women, with a BMI in excess of 25 kg/m(2) to those from women of healthy weight. A Primovision Time-Lapse system was used to measure developmental kinetics and the non-invasive COnsumption/RElese of glucose, pyruvate, amino acids and lactate were measured on spent droplets of culture medium. Total triglyceride levels within individual embryos were also determined. MAIN RESULTS AND THE ROLE OF CHANCE: Human oocytes from women presenting for fertility treatment with a BMI exceeding 25 kg/m(2) are smaller (R(2) = -0.45; P = 0.001) and therefore less likely to complete development post-fertilization (P < 0.001). Those embryos that do develop reach the morula stage faster than embryos from women of a BMI < 25 kg/m(2) (<0.001) and the resulting blastocysts contain fewer cells notably in the trophectoderm (P = 0.01). The resulting blastocysts also have reduced glucose consumption (R(2) = -0.61; P = 0.001), modified amino acid metabolism and increased levels of endogenous triglyceride (t = 4.11, P < 0.001). Our data further indicate that these differences are independent of male BMI. LIMITATIONS, REASONS FOR CAUTION: Although statistical power has been achieved, this is a retrospective study and relatively small due to the scarcity of human embryos available for research. Consequently, subanalysis of overweight and obese was not possible based on the sample size. The analysis has been performed on supernumerary embryos, originating from a single IVF unit and not selected for use in treatment. Thus, it was not possible to speculate how representative the findings would be of the better quality embryos transferred or frozen for each patient. WIDER IMPLICATIONS OF THE FINDINGS: The data indicate that a high BMI of women at conception is associated with distinct phenotypic changes in the embryo during the preimplantation period, highlighting the importance of prepregnancy body weight in optimizing the chances of fertility and safeguarding maternal and offspring health. These changes to the metabolic fingerprint of human embryos which are most likely a legacy of the ovarian conditions under which the oocyte has matured may reduce the chances of conception for overweight women and provide good evidence that the metabolic profile of the early embryo is set by sub-optimal conditions around the time of conception. The observed changes could indicate long-term implications for the health of the offspring of overweight and obese women. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the Hull IVF Unit Charitable Trust and the Hull York Medical School. There are no conflict of interests.

History of oocyte and embryo metabolism.

The basic pattern of metabolism in mammalian oocytes and early embryos was established in the 1960s and 1970s, largely in terms of the consumption of oxygen and the utilisation of nutrients present in culture media at the time, mainly glucose, pyruvate and lactate. The potential importance of endogenous fuels was also recognised but was largely ignored, only to be rediscovered quite recently. The 1980s and 1990s saw the arrival of a 'new generation' of culture media, characterised metabolically by the addition of amino acids, an initiative driven strongly by the need to improve embryo culture and selection methods in assisted reproductive technologies. This trend has continued alongside some basic metabolic studies and the general recognition of the importance of metabolism in all aspects of biology. A framework for future studies on oocyte and early embryo metabolism has been provided by: (1) the developmental origins of health and disease concept and recognition of the relationship between development, epigenetics and metabolism; (2) the need to understand cell signalling within, and between the cells of, the early embryo; and (3) the importance of identifying the mechanisms underlying dialogue between the oocyte and early embryo and the female reproductive tract.

Expression and localization of creatine kinase in the preimplantation embryo.

Creatine Kinase (CK) catalyses the "creatine shuttle," the reversible conversion of creatine phosphate to creatine with the liberation of ATP. This article examines the potential role of the creatine shuttle in the provision of ATP during mouse preimplantation embryo development. Using quantitative PCR, transcripts of four subunit isoforms of CK--CKM, CKB, CKMT1, and CKMT2--were detectable at all developmental stages, from the presumptive zygote to late blastocyst, but there was no obvious pattern in gene expression. By contrast, total CK biochemical activity, measured by a novel method, was relatively constant from the 2- to 8-cell stage, before exhibiting a significant decrease in activity at the blastocyst stage. Immunocytochemical studies revealed a marked association of CKB with the mitotic spindle in 2- and 4-cell mouse embryos, consistent with the proposition that the creatine shuttle plays a key role in local delivery of ATP during cytokinesis. Endogenous creatine was detected in the blastocyst at a level of 0.53 pmol/embryo. In conclusion, we believe that creatine phosphate can now be added to the list of potential sources of ATP during preimplantation development.

Amino acid turnover by bovine oocytes provides an index of oocyte developmental competence in vitro.

Amino acid profiling has been used to distinguish between human embryos of differing developmental competence. We sought to determine whether amino acid profiling could be used to distinguish between metaphase II (MII) bovine oocytes with different developmental capabilities in vitro. Amino acid turnover was assayed during the final 6 h of in vitro maturation prior to oocytes undergoing individual fertilization in vitro. Following insemination, zygotes were immobilized in groups of 16 on the base of a Petri dish using Cell-Tak tissue adhesive to enable the developmental progress of each to be tracked to the blastocyst stage. Spent droplets of in vitro maturation medium were analyzed by high performance liquid chromatography, which revealed glutamine, arginine, and asparagine were depleted in the greatest quantities. Incompetent MII oocytes that failed to cleave by 72 h postfertilization depleted significantly more glutamine from (P = 0.0006) and released more alanine (P = 0.0001) into the medium than oocytes that cleaved. When cutoff values were selected for the turnover of alanine, arginine, glutamine, leucine, and tryptophan and modeled to predict fertilization and cleavage potential, oocytes that did not exceed the cutoff values for ≥2 of these key amino acids were more likely to cleave. The sensitivity, specificity, accuracy, and positive predictive value of this model were 60.5%, 76.8%, 63.5%, and 92.0%, respectively. Significant differences (P ≤ 0.015) in the consumption/production of alanine and glutamine were also observed when comparing uncleaved oocytes with those that produced blastocysts. The data show that noninvasive amino acid profiling can be used to measure oocyte developmental competence.

Metabolism of the preimplantation embryo: 40 years on.

This review considers how our understanding of preimplantation embryo metabolism has progressed since the pioneering work on this topic in the late 1960s and early 1970s. Research has been stimulated by a desire to understand how metabolic events contribute to the development of the zygote into the blastocyst, the need for biomarkers of embryo health with which to improve the success of assisted conception technologies, and latterly by the 'Developmental Origins of Health and Disease' (DOHaD) concept. However, arguably, progress has not been as great as it might have been due to methodological difficulties in working with tiny amounts of tissue and the low priority assigned to fundamental research on fertility and infertility, with developments driven more by technical than scientific advances. Nevertheless, considerable progress has been made in defining the roles of the traditional nutrients: pyruvate, glucose, lactate, and amino acids; originally considered as energy sources and biosynthetic precursors, but now recognized as having multiple, overlapping functions. Other nutrients; notably lipids, are beginning to attract the attention they deserve. The pivotal role of mitochondria in early embryo development and the DOHaD concept, and in providing a cellular focus for metabolic events is now recognized. Some unifying ideas are discussed; namely 'stress-response models' and the 'quiet embryo hypothesis'; the latter aiming to relate the metabolism of individual preimplantation embryos to their subsequent viability. The review concludes by updating the state of knowledge of preimplantation embryo metabolism in the early 1970s and listing some future research questions.

The Quiet Embryo Hypothesis: 20 years on.

This article revisits the hypothesis, proposed in 2002, that the successful development of oocytes and preimplantation mammalian embryos is associated with a metabolism which is "quiet" rather than "active", within limits which had yet to be defined. A distinction was drawn between Functional Quietness, Loss of quietness in response to stress and Inter-individual differences in embryo metabolism and here we document applications of the hypothesis to other areas of reproductive biology. In order to encompass the requirement for "limits" and replace the simple distinction between "quiet" and "active", evidence is presented which led to a re-working of the hypothesis by proposing the existence of an optimal range of metabolic activity, termed a "Goldilocks zone", within which oocytes and embryos with maximum developmental potential will be located. General and specific mechanisms which may underlie the Goldilocks phenomenon are proposed and the added value that may be derived by expressing data on individual embryos as distributions rather than mean values is emphasised especially in the context of the response of early embryos to stress and to the concept of the Developmental Origins of Health and Disease. The article concludes with a cautionary note that being "quietly efficient" may not always ensure optimal embryo survival.

Amino Acids and the Early Mammalian Embryo: Origin, Fate, Function and Life-Long Legacy.

Amino acids are now recognised as having multiple cellular functions in addition to their traditional role as constituents of proteins. This is well-illustrated in the early mammalian embryo where amino acids are now known to be involved in intermediary metabolism, as energy substrates, in signal transduction, osmoregulation and as intermediaries in numerous pathways which involve nitrogen metabolism, e.g., the biosynthesis of purines, pyrimidines, creatine and glutathione. The amino acid derivative S-adenosylmethionine has emerged as a universal methylating agent with a fundamental role in epigenetic regulation. Amino acids are now added routinely to preimplantation embryo culture media. This review examines the routes by which amino acids are supplied to the early embryo, focusing on the role of the oviduct epithelium, followed by an outline of their general fate and function within the embryo. Functions specific to individual amino acids are then considered. The importance of amino acids during the preimplantation period for maternal health and that of the conceptus long term, which has come from the developmental origins of health and disease concept of David Barker, is discussed and the review concludes by considering the potential utility of amino acid profiles as diagnostic of embryo health.

Association between amino acid turnover and chromosome aneuploidy during human preimplantation embryo development in vitro.

This study investigated the relationship between human preimplantation embryo metabolism and aneuploidy rates during development in vitro. One hundred and eighty-eight fresh and cryopreserved embryos from 59 patients (33.9 +/- 0.6 years) were cultured for 2-5 days. The turnover of 18 amino acids was measured in spent media by high-performance liquid chromatography. Embryos were either fixed for interphase fluorescent in situ hybridization analysis of chromosomes 13, 18, 19, 21, X or Y, or were assayed for mitochondrial activity. Amino acid turnover was different (P < 0.05) between stage-matched fresh and cryopreserved embryos due to blastomere loss following warming. The proportion of embryos with aneuploid cells increased as cell division progressed from pronucleate- (23%) to late cleavage stages (50-70%). Asparagine, glycine and valine turnover was significantly different between uniformly genetically normal and uniformly abnormal embryos on Days 2-3 of culture. By Days 3-4, the profiles of serine, leucine and lysine differed between uniformly euploid versus aneuploid embryos. Gender significantly (P < 0.05) affected the metabolism of tryptophan, leucine and asparagine by cleavage-stage embryos. Pronucleate zygotes had a significantly higher proportion of active:inactive mitochondria compared with cleavage-stage embryos. Furthermore, mitochondrial activity was correlated (P < 0.05) with altered aspartate and glutamine turnover. These results demonstrate the association between the metabolism, cytogenetic composition and health of human embryos in vitro.

DNA damage and metabolic activity in the preimplantation embryo.

BACKGROUND: Embryos with greater viability have a lower or 'quieter' amino acid metabolism than those which arrest. We have hypothesized this is due to non-viable embryos possessing greater cellular/molecular damage and consuming more nutrients, such as amino acids for repair processes. We have tested this proposition by measuring physical damage to DNA in bovine, porcine and human embryos at the blastocyst stage and relating the data to amino acid profiles during embryo development. METHODS: Amino acid profiles of in vitro-derived porcine and bovine blastocysts were measured by high-performance liquid chromatography and the data related retrospectively to DNA damage in each individual blastomere using a modified alkaline comet assay. Amino acid profiles of spare human embryos on Day 2-3 were related to DNA damage at the blastocyst stage. RESULTS: A positive correlation between amino acid turnover and DNA damage was apparent when each embryo was examined individually; a relationship exhibited by all three species. There was no relationship between DNA damage and embryo grade. CONCLUSIONS: Amino acid profiling of single embryos can provide a non-invasive marker of DNA damage at the blastocyst stage. The data are consistent with the quiet embryo hypothesis with viable embryos (lowest DNA damage) having the lowest amino acid turnover. Moreover, these data support the notion that metabolic profiling, in terms of amino acids, might be used to select single embryos for transfer in clinical IVF.

Pyruvate and oxygen consumption throughout the growth and development of murine oocytes.

Growing oocytes in vitro from the most immature stages until they are developmentally competent is a major goal of reproductive technology, requiring fundamental knowledge of metabolic processes. Carbohydrate metabolism and oxygen consumption have been analysed in a series of experiments designed to investigate important energy substrates for mouse oocytes and to reveal any qualitative or quantitative changes between the primordial and ovulatory follicle stages. Primordial follicles were incubated in groups in modified-KSOM medium, whereas growing or ovulated oocytes were studied singly and, in both cases, the depletion or accumulation of metabolites in spent medium were analysed using ultramicrofluorometric assays. The rates of glucose (0.014 +/- 0.006 pmol/hr) and pyruvate (0.028 +/- 0.009 pmol/hr) consumption and l-lactate (0.058 +/- 0.023 pmol/hr) production by primordial follicles suggested that energy production was supported by a combination of metabolic pathways, including glycolysis. Pyruvate and oxygen consumption per oocyte increased two- and ninefold, respectively, between the primary and pre-ovulatory stages (0.82 +/- 0.1 and 1.67 +/- 0.1 pmol pyruvate/hr, respectively and 1.4 +/- 0.3 and 7 +/- 0.6 pmol oxygen/hr) after which oxygen (12.7 +/- 1.1 pmol/hr) utilisation nearly doubled. Oxygen consumption by fully grown oocytes was in excess of oxidation requirements for pyruvate. When pyruvate and oxygen consumption rates were normalised for oocyte cellular volume, which increased over 130-fold during growth, oocyte metabolism was higher in primary follicles than at any subsequent stage, indicating that energy needs are greater during a developmental transition. To conclude, pyruvate and oxygen were consumed throughout oocyte development at increasing rates. When oocyte cellular volume was accounted for, oocytes from primary follicles displayed greatest metabolic rates.

Going to extremes: the Goldilocks/Lagom principle and data distribution.

Numerical data in biology and medicine are commonly presented as mean or median with error or confidence limits, to the exclusion of individual values. Analysis of our own and others' data indicates that this practice risks excluding 'Goldilocks' effects in which a biological variable falls within a range between 'too much' and 'too little' with a region between where its function is 'just right'; a concept captured by the Swedish term 'Lagom'. This was confirmed by a narrative search of the literature using the PubMed database, which revealed numerous relationships of biological and clinical phenomena of the Goldilocks/Lagom form including quantitative and qualitative examples from the health and social sciences. Some possible mechanisms underlying these phenomena are considered. We conclude that retrospective analysis of existing data will most likely reveal a vast number of such distributions to the benefit of medical understanding and clinical care and that a transparent approach of presenting each value within a dataset individually should be adopted to ensure a more complete evaluation of research studies in future.

Application of extracellular flux analysis for determining mitochondrial function in mammalian oocytes and early embryos.

Mitochondria provide the major source of ATP for mammalian oocyte maturation and early embryo development. Oxygen Consumption Rate (OCR) is an established measure of mitochondrial function. OCR by mammalian oocytes and embryos has generally been restricted to overall uptake and detailed understanding of the components of OCR dedicated to specific molecular events remains lacking. Here, extracellular flux analysis (EFA) was applied to small groups of bovine, equine, mouse and human oocytes and bovine early embryos to measure OCR and its components. Using EFA, we report the changes in mitochondrial activity during the processes of oocyte maturation, fertilisation, and pre-implantation development to blastocyst stage in response to physiological demands in mammalian embryos. Crucially, we describe the real time partitioning of overall OCR to spare capacity, proton leak, non-mitochondrial and coupled respiration - showing that while activity changes over the course of development in response to physiological demand, the overall efficiency is unchanged. EFA is shown to be able to measure mitochondrial function in small groups of mammalian oocytes and embryos in a manner which is robust, rapid and easy to use. EFA is non-invasive and allows real-time determination of the impact of compounds on OCR, facilitating an assessment of the components of mitochondrial activity. This provides proof-of-concept for EFA as an accessible system with which to study mammalian oocyte and embryo metabolism.

Metabolism of the viable mammalian embryo: quietness revisited.

This review examines the 'Quiet Embryo Hypothesis' which proposes that viable preimplantation embryos operate at metabolite or nutrient turnover rates distributed within lower ranges than those of their less viable counterparts. The 'quieter' metabolism consistent with this hypothesis is considered in terms of (i) 'functional' quietness; the contrasting levels of intrinsic metabolic activity in different cell types as a consequence of their specialized functions, (ii) inter-individual embryo/cell differences in metabolism and (iii) loss of quietness in response to environmental stress. Data are reviewed which indicate that gametes and early embryos function in vivo at a lower temperature than core body temperature, which could encourage the expression of a quiet metabolism. We call for research to determine the optimum temperature for mammalian gamete/embryo culture. The review concludes by examining the key role of reactive oxygen species, which can induce molecular damage, trigger a cellular stress response and lead to a loss of quietness.

Symposium: innovative techniques in human embryo viability assessment. Assessing embryo viability by measurement of amino acid turnover.

This review assesses the ability of non-invasive 'amino acid profiling' to predict early embryo viability. The history of amino acid supplementation of embryo culture media and the role of amino acids in early embryo development are first considered and these are followed by a review of methods to quantify amino acid depletion and production by single embryos. Data on amino acid profiling of embryos from a number of species are then discussed. It is concluded that this technology has excellent potential to improve the selection of single embryos for transfer in clinical IVF.

Female reproductive tract fluids: composition, mechanism of formation and potential role in the developmental origins of health and disease.

The oviduct and uterus provide the environments for the earliest stages of mammalian embryo development. However, little is known about the mechanisms that underlie the formation of oviduct and uterine fluids, or the extent to which the supply of nutrients via these reproductive tract tissues matches the nutrient requirements of early embryos. After reviewing our limited knowledge of these phenomena, a new experimental paradigm is proposed in which the epithelia lining the endosalpinx and endometrium are seen as the final components in a supply line that links maternal diet at one end and embryo uptake of nutrients at the other. When considered in this way, the oviduct and uterine epithelia become, for a few days, potentially the most critical maternal tissues in the establishment of a healthy pregnancy. In fulfilling this 'gatekeeper' role, female reproductive tract fluids have a key role in the 'developmental origins of health and disease' concept.

Expression and function of transient receptor potential channels in the female bovine reproductive tract.

The epithelium lining the oviduct is critical for early reproductive events, many of which are mediated via intracellular calcium ions. Despite this, little is known about the regulation of calcium homeostasis in the oviductal epithelium. Epithelial transient receptor potential channels (TRPCs) modulate calcium flux in other tissues, and their expression and functional regulation have therefore been examined using the bovine oviduct as a model for the human. The effects of FSH, LH, 17ß-estradiol, and progesterone on TRPCs expression and intracellular calcium flux were determined. Transient receptor potential channels 1, 2, 3, 4, and 6 were expressed in the bovine reproductive tract, and their gene expression varied throughout the estrous cycle. In more detailed studies undertaken on TRPC1 and 6, we show that protein expression varied through the estrus cycle; specifically, 17ß-estradiol, FSH, and LH individually and in combination upregulated TRPC1 and 6 expression in cultured bovine oviduct epithelial cells although progesterone antagonized these effects. Functional studies showed changes in calcium mobilization in bovine oviduct epithelial cells were dependent on TRPCs. In conclusion, TRPC1, 2, 3, 4, and 6 are present in the epithelium lining the bovine oviduct, and TRPC1 and 6 vary through the estrous cycle suggesting an important role in early reproductive function.