Fundamental cryobiology of reproductive cells and tissues

doi:10.1016/j.cryobiol.2004.03.002

Cryobiology

Volume 48, Issue 2, April 2004, Pages 146-156

https://doi.org/10.1016/j.cryobiol.2004.03.002 Get rights and content

Abstract

During the last half of the 20th century there have been considerable advancements in mammalian reproductive technologies, including in vitro production of pre-implantation embryos and embryo sexing, and even cloning in some species. However, in most cases, management of non-cryopreserved reproductive cells (i.e., spermatozoa or oocytes) and tissues (i.e., testicular tissue or ovarian tissue) is problematic due to difficulties in donor–recipient synchronization and the potential for transmission of infectious pathogens, which cumulatively limits widespread application of these techniques. Therefore, there is an urgent need for the development of optimum cryopreservation methods for reproductive cells and tissues from many species. Today frozen–thawed spermatozoa and embryos have become an integral component of animal agriculture, laboratory animal genome banking, and human sperm banking and infertility programs. However, although widely implemented, the protocols currently used to cryopreserve bull sperm, for example, are still suboptimal, and cannot readily be extrapolated to other species’ sperm. Similarly, embryo-freezing protocols successfully used for mouse and cattle have yielded little success when applied to some other species’ embryos, or to a related cell type, oocytes. To date, with the exception of mouse oocytes, almost all mammalian species’ oocytes studied have proven very difficult to successfully cryopreserve. Currently, there is a growing interest to understand the underlying cryobiological fundamentals responsible for these low survival rates in an effort to develop better cryopreservation methods for oocytes. Additionally, there is growing interest in developing technologies for the optimal isolation and cryopreservation of the earliest stage of male (spermatogonia, spermatids) and female (primordial follicle) germ cells, with subsequent maturation to the desired stage in vitro. Female gamete maturation, fertilization, and embryo development entirely under in vitro conditions from primordial follicles has been achieved in mice, however techniques for this and other species are still very early in their development. Furthermore, with the recent advances made in intracytoplasmic sperm injection (ICSI), and gamete isolation and maturation, close attention has been given to cryopreservation of gametes in the form of gonadal tissue (i.e., testicular tissue and ovarian tissue) containing various developmental stages of male (spermatogonia, spermatids, and spermatozoa) and female (primordial, secondary) germ lines.

Section snippets

Fundamental cryobiology

In the context of this report, “fundamental cryobiology” refers to understanding the effects of low temperature on cellular systems, and utilizing this knowledge to develop improved cryopreservation procedures. Historically, this has involved the interdisciplinary research of biologists, chemists, physicists, engineers, and others using carefully designed empirical evaluations, as well as investigation and utilization of specific cellular characteristics, in efforts to apply cryopreservation to

Spermatozoa

The relative success achieved with semen cryopreservation has allowed significant advancements in the fields of agriculture, by making available an international exchange of germplasm of genetically superior animals; biotechnology, by allowing scientifically important murine lines to be efficiently stored; conservation of endangered species through genome resource banking; and human reproductive medicine.

While there have been some recent, exciting studies involving freeze-drying of mouse sperm

Testicular tissue

Currently there is an increasing research effort directed towards the utilization of cryopreserved testicular tissue containing abundant numbers of germ cells at various developmental stages [63]. Advanced stage (i.e., spermatids, spermatozoa) germ cells can be successfully retrieved from cryopreserved testicular tissue [8], [69] following mechanical extraction or enzymatic digestion, and it is now possible to harvest these cells at earlier stages of development (i.e., spermatogonia) for

Conclusion

The ability to successfully preserve and store reproductive cells and tissues has significant importance in the advancement of assisted reproductive technology and medicine. The progress in preservation of this material is enabling better management of livestock and laboratory animal species, better conservation of biodiversity, and improved modalities for the treatment of human infertility. The technology behind reproductive cell and tissue cryopreservation has been derived from basic

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Cited by (221)

Extension of the equilibration period up to 24 h maintains the post-thawing quality of Holstein bull semen frozen with OPTIXcell®
2023, Animal Reproduction Science
Semen cryopreservation in bovine livestock is well established, but logistics often require deviations from standard protocols. Extending the equilibration time to the following day is convenient in many situations. To improve our knowledge of the effects of this modification, we studied the post-thawing and post-incubation (4 h, 38 °C) sperm quality after freezing with 4 or 24-h extension in the OPTIXcell extender by using an ample panel of analyses: CASA for motility; flow cytometry for viability, physiology, oxidative stress, and chromatin parameters (DNA fragmentation, chromatin compaction, and thiol groups status); and spectrometry for malondialdehyde production. Semen was obtained from 12 Holstein bulls. The 24-h equilibration time showed few significant effects, with only a tiny decrease in progressive motility and a positive impact on chromatin structure. The incubation removed some of these effects, with the pattern for chromatin compaction remaining the same. No detrimental oxidative stress or increase in apoptotic or capacitation markers was detected. Additionally, the individual bull interacted with the effects of the incubation and the equilibration, especially regarding the chromatin status. Whereas this interaction did not critically affect sperm quality, it could be relevant in practice. Bull fertility as non-return rates (NRR56) was associated with some sperm parameters (especially with an improved chromatin structure) but not in the 4-h post-thawing analysis. Our study supports that extending the equilibration time by at least 24-h is feasible for bull semen freezing with the OPTIXcell extender.
New insight into the mechanism by which antifreeze peptides regulate the physiological function of Streptococcus thermophilus subjected to freezing stress
2023, Journal of Advanced Research
Citation Excerpt :
However, cryopreservation agents will be inevitably used for the development of lyophilized oral vaccines when the cells need to be freeze-dried. In fact, cryopreservation of living cells and tissues is fundamental in modern biology, food, agriculture and medicinal research [2]. Lactic acid bacteria (LAB) are important microorganisms in the modern biological and food industries.
Antifreeze peptides regulate the physiological functions of frozen cells and even their apoptosis; however, the mechanisms by which antifreeze peptides regulate these processes remain unclear, although the interactions between cell membranes and ice are well known to be important in this process.
Our study aims to investigate how antifreeze peptides regulate cell physiological functions during the freezing process.
We investigated the cryoprotective effect of rsfAFP on the physiological functions of S. thermophilus under freezing stress by measuring cellular metabolism activity, intracellular enzyme activity, cell membrane characterization, and cell apoptosis. The mechanism by which rsfAFP impacts S. thermophilus physiological functions under freezing stress was investigated using multispectral techniques and cryo-TEM.
We show that a recombinant antifreeze peptide (rsfAFP) interacts with the extracellular capsular polysaccharides and peptidoglycan of Streptococcus thermophilus and ice to cover the outer layer of the membrane, forming a dense protective layer that regulates the molecular structure of extracellular ice crystals, which results in reduced extracellular membrane damage, depressed apoptosis and increased intracellular metabolic activity. This interaction mechanism was indicated by the fact that S. thermophilus better maintained its permeability barrier, membrane fluidity, membrane structural integrity, and cytoplasmic membrane potential during freezing stress with rsfAFP treatment.
These results provide new insights into the mechanism by which rsfAFP regulates frozen cell physiological functions and apoptosis under freezing stress.
Real-time computer assisted measurement of oocyte and embryo volume for assessment of transport parameters
2022, Cryobiology
Cryopreservation of gametes has revolutionized both animal agriculture and human reproductive medicine. Although many new technologies have tremendously improved the cryopreservation of oocytes and embryos, osmotic stress encountered during the equilibration process can cause their loss of function. Rational cryoprotective agent (CPA) equilibration strategies can be used to minimize this stress but require trained personnel to monitor the process in individual oocytes or embryos or require the use of suboptimal average transport parameter values in mathematically guided protocols. To enable individually optimized equilibration of CPAs in individual cells, here we establish experimental and computational techniques to track the osmotic behavior of individual bovine oocytes and embryos during CPA equilibration in real time. We designed a microfluidic device to provide a controlled flow of CPA and modified standard image analysis techniques to estimate real-time cell volume changes. In particular, we used a level-set method to define a boundary within a contour plot which could automate the image analysis process. A colour based level set algorithm coupled with contour smoothing not only provided the best fit but also reduced the segmentation time to well under a second per image. The accuracy of the automated method was comparable to human segmented images for both oocytes and embryos. This technology should enable both rapid evaluation of key biophysical parameters in oocytes and embryos undergoing CPA equilibration and the development of real-time feedback-control of CPA equilibration, enabling individual oocyte- and embryo-specific optimal protocols.
Sperm freezability is neither associated with the expression of aquaporin 3 nor sperm head dimensions in dromedary camel (Camelus dromedarius)
2022, Theriogenology
The expression of aquaglyceroporin 3 (AQP-3) has been demonstrated in the spermatozoa of several mammalian species and its role has been associated with cryotolerance. Post-thaw sperm quality from individual dromedary males with different response to freezing-thawing process was evaluated through sperm head morphometry. In order to understand the cellular mechanisms affected by cryoinjury we have explored the presence and distribution of sperm AQP-3 using western blotting and immunocytochemistry. WB showed different intensity of the specific signal bands at 28 kDa. Immunofluorescence assessments allowed us to identify five different and clear AQP-3 distribution patterns of labelling in the sperm plasma membrane; acrosome, post-acrosome, mid-piece, and principal and final tail. Although expression of AQP-3 varied among male ejaculates, the individual sperm response to freeze-thawing was not associated with AQP-3 expression. Thus, AQP3 expressions do not seem like a reliable predictor of sperm response to freeze-thawing process in this species. This work is the first to describe the morphometric characteristics of the heads of dromedary spermatozoa. No correlation was found between sperm head dimensions and sperm quality variables after freeze-thawing suggesting that dromedary camel sperm head morphometry is also not a reliable predictor of cryosurvival.
Diluent Containing Dimethylformamide Added With Sucrose Improves In Vitro Quality After Freezing/Thawing Stallion Sperm
2022, Journal of Equine Veterinary Science
The aim of this study was to investigate the effects of sucrose on post-thawed equine semen quality. Semen samples (n = 24) were collected from six stallions. They were diluted (200 × 10⁶ sperm/mL) in a freezing medium based on skimmed milk, egg yolk, dimethylformamide, and supplemented with sucrose at concentrations of 0 (Control), 25, 50, and 100 mM and in a commercial extender (BotuCrio). Subsequently, they were filled in straws (0.5 mL) and subjected to freezing and storage (−196°C). Immediately after thawing (37°C, 30 seconds), semen samples were evaluated for kinetics (CASA), plasma and acrosomal membrane integrity, and mitochondrial membrane potential (flow cytometry). The addition of 50 and 100mM sucrose to the freezing extender increased (P < .05) the parameters of TM, PM, VCL, VSL, and VAP, compared to the control group. The WOB parameter of the group supplemented with 100 mM sucrose was higher (P < .05) than the control group. Higher values (P < .05) of ALH and BCF were observed in groups treated with sucrose (25, 50, and 100 mM), compared to BotuCrio. The semen frozen in the presence of 100 mM sucrose presented higher percentages (P < .05) of sperm with intact plasma and acrosomal membranes, and high mitochondrial membrane potential in relation to the other groups. It is concluded that the addition of sucrose to equine semen freezing extender increase motility (50 and 100 mM), plasma and acrosomal membrane integrity preserve, and high sperm mitochondrial membrane potential (100 mM) after thawing.
Quercetin effect on the efficiency of ovine oocyte vitrification at GV stage
2021, Theriogenology
The widely adopted method of vitrification is known to induce some negative effects on oocytes. In order to enhance the efficiency of this process performed on ovine oocytes at germinal vesicle stage, vitrification and warming (VW) solutions, and maturation media were supplemented with 5 μM Quercetin (Q). Four groups of vitrified and fresh immature oocytes were subjected to IVM, IVF and IVC, and their survival rate, apoptosis, nuclear status and developmental competence were assessed. Non-vitrified oocytes treated with Quercetin experienced higher cleavage rate relative to those matured without Quercetin (p < 0.05). Supplementation of VW and maturation media with Quercetin resulted in increased survival, cleavage and total blastocyst rates relative to the untreated oocytes. The post-IVM survival rate of non-vitrified oocytes showed no difference among those matured with and without Quercetin, but was higher for oocytes vitrified, warmed and matured with Quercetin relative to VW group lacking Quercetin. The proportion of early-apoptotic (AV+) oocytes was affected by Quercetin supplementation in both control and VW groups (p < 0.05). The number of AV positive oocytes was lower and the proportion of oocytes reaching MII stage was greater in non-vitrified and VW groups matured with Quercetin, in comparison with their untreated respective controls (p < 0.05). There was no difference in the number of late-stage apoptotic oocytes among different groups. It is concluded that supplementing vitrification and warming solutions with Quercetin endows vitrified ovine oocytes with protective potential against early apoptotic damage, and improves viability, maturation rate and developmental competence at GV stage.

View all citing articles on Scopus

^☆: This work was funded by the University of Missouri Center for Comparative Medicine, grants from the National Institutes of Health (R24 RR13194, U42 RR1482, and R24 RR153770).

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Fundamental cryobiology of reproductive cells and tissues☆

Abstract

Section snippets

Fundamental cryobiology

Spermatozoa

Testicular tissue

Conclusion

Cryobiology

Cryobiology

Mol. Cell. Endocrinol.

Cryobiology

Fertil. Steril.

Theriogenology

Biomaterials

Cryobiology

Theriogenology

Cryobiology

Cryobiology

Cryobiology

Theriogenology

Cryobiology

Fertil. Steril.

Cryobiology

Fertil. Steril.

Fertil. Steril.

Fertil. Steril.

Osmotic characteristics of mouse spermatozoa in the presence of extenders and sugars

Biol. Reprod.

Effect of osmotic stress on the developmental competence of germinal vesicle and metaphase II stage bovine cumulus oocyte complexes and its relevance to cryopreservation

Mol. Reprod. Dev.

Cryopreservation of oocytes and ovarian tissue

ILAR J.

Effects of cooling and rewarming on the meiotic spindle and chromosomes of in vitro-matured bovine oocyte

Biol. Reprod.

Reconstitution of spermatogenesis from frozen spermatogonial stem cells

Nat. Med.

Testicular tissue cryopreservation in boys, ethical and legal issues

Hum. Reprod.

Freezing of testicular tissue as a minced suspension preserves sperm quality better than whole-biopsy freezing when glycerol is used as cryoprotectant

Int. J. Androl.

Cryopreservation of mature and immature gametes

Cryopreservation of murine spermatozoa

ILAR J.

Calculated optimal cooling rates for ram and human sperm cryopreservation fail to conform with empirical results

Biol. Reprod.

Immature rat ovaries grafted after freezing and thawing

J. Endocrinol.

An irreversible thermodynamic analysis of cell freezing in the presence of membrane permeable additives. II. Transient electrolyte and additive concentrations

Cryobiology

Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol

Hum. Reprod.

Use of fetal bovine serum substitutes for the protection of the mouse zona pellucida against hardening during cryoprotectant addition

Hum. Reprod.

Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa

Hum. Reprod.

Osmotic tolerance limits of murine spermatozoa in the presence of extender media

Cryobiology

Cryoprotective agent and temperature effects on human sperm membrane permeabilities: convergence of theoretical and empirical approaches for optimal cryopreservation methods

Hum. Reprod.

Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa

Hum. Reprod.

Preservation of fertility in nature and ART

Reproduction

Utility of viable tissues ex vivo

Allogeneic and xenogeneic transplantation of cryopreserved ovarian tissue to athymic mice

Biol. Reprod.

Sperm from neonatal mammalian testes grafted in mice

Nature