In vitro growth, maturation, fertilization, and embryonic development of oocytes from porcine preantral follicles

This study was conducted to identify an in vitro culture system that would support intact porcine follicle growth from preantral follicle to antral stages, oocyte maturation, fertilization, and embryonic development; and to evaluate factors that influence porcine preantral follicle growth in vitro. Preantral follicles isolated from prepubertal porcine ovaries were cultured for 4 days in the presence of different concentrations of porcine serum and FSH, and with different numbers of follicles per well. A series of experiments showed that porcine antral follicles can be grown at a high frequency in vitro from healthy preantral follicles with intact theca when cultured in North Carolina State University 23 medium supplemented with 1.5 ng/ml FSH, 7.5% serum, and when cultured with three follicles per well. After 4 days of culture, 68% healthy cumulus-enclosed oocytes from these follicles were obtained, and 51% of the oocytes completed meiotic maturation to the metaphase II stage. Fifty-three percent of the mature oocytes underwent fertilization, 43% of the fertilized oocytes cleaved, and 13% developed to the blastocyst stage. The results show 1) that porcine preantral follicles can grow efficiently to the antral stage using these culture conditions, and 2) that oocytes from in vitro-matured porcine preantral follicles can acquire meiotic competence and undergo fertilization and embryonic development.     

Role of follicle stimulating hormone and epidermal growth factor in the development of porcine preantral follicle in vitro

The aim of the present study was to assess the role of follicle stimulating hormone (FSH), epidermal growth factor (EGF) or a combination of EGF and FSH on the in vitro growth of porcine preantral follicles, estradiol secretion, antrum formation, oocyte maturation and subsequent embryonic development. Porcine preantral follicles were cultured for 3 days in the absence or in the presence of FSH or EGF. Oocytes from these follicles were then matured, fertilized in vitro and embryos were cultured. Estradiol secretion and histological analysis of cultured follicles were also carried out. The results showed that when FSH, or a combination of EGF and FSH, was added to the culture medium, most of preantral follicles grew to antral follicles with high estradiol secretion and the oocytes from these antral follicles could mature, fertilize and develop to the blastocyst stage. Without FSH, or a combination of EGF and FSH, preantral follicles were unable to develop to the antral stage. Histology demonstrated that the resulting follicles were nonantral, estradiol production was reduced and none of their oocytes matured after in vitro maturation. The results indicate the essential role of FSH in promoting in vitro growth of porcine preantral follicle, estradiol secretion, antrum formation, oocyte maturation and subsequent embryonic development. EGF with FSH treatment of porcine preantral follicles improves the quality of oocytes, shown by a higher frequency of embryonic development.     

Mouse oocytes derived from fetal germ cells are competent to support the development of embryos by in vitro fertilization

Mouse oocyte development in vitro has been studied in the past several years, but no evidence showed that the fertilizable oocytes could be obtained from the fetal mouse germ cells before the formation of the primordial follicles. In this study, an efficient and simple method has been established to obtain the mature oocytes from the fetal mouse germ cells at 16.5 days post-coitum (dpc). For the initial of follicular formation, fetal mouse 16.5 dpc ovaries were transplanted to the recipient under the kidney capsule, and the ovaries were recovered after 14 days. Subsequently, the growing preantral follicles in the ovarian grafts were isolated and cultured in vitro for 12 days. Practically, the mature oocytes ovulated from the antral follicles were able to be fertilized in vitro and support the embryonic development. The results demonstrate that the fetal mouse 16.5 dpc germ cells are able to form primordial follicles with the ovarian pregranulosa cells during the period of transplantation in the ectopic site, and the oocytes within the growing follicles are able to mature in vitro, then are able to support the embryonic development.     

Development of in vitro-matured oocytes from porcine preantral follicles following intracytoplasmic sperm injection.

The objective of this study was to assess fertilization and embryonic development following intracytoplasmic sperm injection (ICSI) of oocytes from porcine preantral follicles matured in vitro. Also, another aim was to describe actin filament distribution during fertilization and embryonic development of those oocytes after ICSI as one of the factors assessed. Preantral follicles isolated from prepubertal porcine ovaries were cultured in a system that supports follicular development. After in vitro maturation, the oocytes were fertilized by ICSI or conventional fertilization in vitro (IVF). Actin filaments of the fertilized oocytes and embryos produced by ICSI or IVF were stained by rhodamine-phalloidin and visualized by fluorescence microscopy. ICSI resulted in 64% fertilization of porcine preantral follicle oocytes matured in vitro. Of those, 51% of the fertilized oocytes cleaved and 21% developed to the blastocyst stage. No significant differences in percentages of oocyte fertilization, cleavage, and blastocyst formation were observed between ICSI and IVF (53%, 45% and 16%, respectively). Actin filament distribution during fertilization and embryonic development of ICSI- or IVF-fertilized oocytes from porcine preantral follicles was similar to that of oocytes derived from antral follicles and fertilized by standard IVF. These results indicate that oocytes from porcine preantral follicles matured in vitro following ICSI can undergo fertilization and subsequent embryonic development.     

Recent achievements in in vitro culture and preservation of ovarian follicles in mammals

The mammalian ovary contains a large number of follicles that are in various developmental stages. The largest portion of them are primordial follicles. However, throughout the female reproductive lifespan only a small proportion of these follicles will produce oocytes competent to undergo successful maturation and ovulation. The rest of the ovarian oocytes (>99.9%) undergo atresia. It would be of great practical benefit to rescue some of these follicles by growing them in culture in order to provide an extra source of gametes. There is considerable interest in developing technologies that aim to produce fully-grown, developmentally competent oocytes from a pool of early developmental stages of follicles. Two methods have been used: 1/ long-term in vitro culture of either follicles or oocytes, and 2/ transplantation of ovarian tissue grafts. The development of efficient technologies may provide an additional source of oocytes for livestock production and reproduction in humans and rare or endangered species. The aim of this paper is to present a comprehensive review of recent achievements in the utilization of small ovarian follicles (primordial, preantral and early antral) by long-term in vitro culture and/or transplantation of ovarian tissue grafts (fresh and cryopreserved) in mammals including humans.     

Maturation of mouse primordial follicles by combination of grafting and in vitro culture

Cryopreservation of ovarian cortical tissue and subsequent transplantation or in vitro culture of follicles are technologies under development with the aim to safeguard fertility in patients with gonadal failure. In the present study, we investigated whether primordial follicles could be triggered to full maturation by a combination of in vivo transplantation and in vitro culture in a mouse model. In a first step, newborn mouse ovaries containing only primordial follicles were allotransplanted under the renal capsule of ovariectomized recipient mice. The second step was to mechanically isolate growing preantral follicles from the graft and culture these in vitro to maturity. In our experiment, one newborn mouse ovary was transplanted under the renal capsule of each 8- to 12-wk-old F1 (C57Bl/6j x CBA/Ca) female ovariectomized recipient (n = 26). Two weeks after transplantation, all 26 grafts were recovered. Four grafts were processed for histology and showed that developmental stages of follicles in 14-day-old ovarian grafts were comparable to those in 14-day-old mouse ovaries. The 22 remaining grafts were used for mechanical isolation of preantral follicles. As a control group, preantral follicles isolated from ovaries of 14-day-old mice were used. The mean preantral follicle yield per ovary was 11 in the transplant group versus 33 in the control group. Follicles were cultured individually in 20-microliter droplets of alpha-MEM supplemented with 100 mIU rFSH and 5% fetal bovine serum for 12 days under an atmosphere of 5% CO(2) in air at 37 degrees C. By Day 12 of culture, 66.5% of follicles retained their oocytes in the grafting group versus 97.5% in the control group (P < 0.001). Final oocyte maturation was induced by addition of 2.5 IU/ml hCG. At 14-16 h post-HCG, the percentages of oocytes showing germinal vesicle breakdown and polar body extrusion were significantly higher in the control group (90.6% and 82.8%) compared to the grafting group (60% and 45%). The mean diameter of the mature oocytes of the grafting group (69.9 +/- 4.45 micrometer) was similar to that of oocytes from the control group (70.5 +/- 2.35 micrometer). Our results suggest that maturation of mouse primordial follicles is feasible by combination of in vivo transplantation and in vitro culture. This two-step strategy may be an attractive model for promoting the growth and maturation of primordial follicles from other species.     

Mammalian oocyte growth and development in vitro

This paper is a review of the current status of technology for mammalian oocyte growth and development in vitro. It compares and contrasts the characteristics of the various culture systems that have been devised for the culture of either isolated preantral follicles or the oocyte-granulosa cell complexes from preantral follicles. The advantages and disadvantages of these various systems are discussed. Endpoints for the evaluation of oocyte development in vitro, including oocyte maturation and embryogenesis, are described. Considerations for the improvement of the culture systems are also presented. These include discussions of the possible effects of apoptosis and inappropriate differentiation of oocyte-associated granulosa cells on oocyte development. Finally, the potential applications of the technology for oocyte growth and development in vitro are discussed. For example, studies of oocyte development in vitro could help to identify specific molecules produced during oocyte development that are essential for normal early embryogenesis and perhaps recognize defects leading to infertility or abnormalities in embryonic development. Moreover, the culture systems may provide the methods necessary to enlarge the populations of valuable agricultural, pharmaceutical product-producing, and endangered animals, and to rescue the oocytes of women about to undergo clinical procedures that place oocytes at risk. © 1996 Wiley-Liss, Inc.     

Activation of follicle development: the primordial follicle

Investigations of primordial follicle formation and growth are fundamental to our understanding of female gamete production. In all mammalian females the full complement of oocytes is established during fetal development. This store of primordial follicles is not renewable and serves the entire reproductive life span of the adult. The correct programming of fetal ovarian development and the number of primordial follicles formed will therefore limit the fecundity of the ovary. Primordial follicles are characterized by the presence of a single oocyte surrounded by a varying number of pregranulosa cells. The relatively small size, undifferentiated status and large numbers of primordial follicles make them prime candidates for use in basic and applied research in animal production, gene transfer and cloning. Furthermore, the development of cell culture systems that use primordial follicles as a source of oocytes for in vitro growth and maturation will enable us to maximize the potential of high genetic merit females and to shorten generation intervals. Despite these possibilities, primordial follicles are the least understood of all stages of follicle development. The factor(s) responsible for maintaining the primordial pool or, conversely, for activating primordial follicle growth remain elusive.     

Kit ligand and glial‐derived neurotrophic factor as alternative supplements for activation and development of ovine preantral follicles in vitro

In vitro growth of preantral follicles has the potential to produce considerable numbers of competent oocytes for use in medicine, agriculture, and even wildlife conservation. The critical regulatory role of growth factors and hormones in the development of preantral follicles has been established. This study investigated the effect of glial‐derived neurotropic factor (GDNF) and kit ligand (KL) on the in vitro development of ovine preantral follicles. Results indicated that both GDNF and KL significantly improved activation of primordial follicles, similar to co‐addition of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), which are commonly used for in vitro follicular development. Importantly, GDNF had a more profound effect on follicle health, development, and differentiation compared with KL alone. Furthermore, the combination of GDNF and KL in the presence of EGF and bFGF had a positive, synergic effect on health, development, and differentiation of preantral follicles, as determined by histological and hormonal assessments. The results of this study may provide a foundation for further studies that will unravel the molecular mechanisms of follicular development to further improve the current status of in vitro preantral follicle culture. Mol. Reprod. Dev. 80: 35–47, 2013. © 2012 Wiley Periodicals, Inc.     

The early stages of follicular development: activation of primordial follicles and growth of preantral follicles

Although enormous progress has been made in understanding the events and regulation of the later stages of ovarian follicular development, the early stages of development, to a large extent and particularly in large mammals, remain a mystery. Mechanisms that regulate the initiation of follicular growth (follicle activation) and the ensuing growth and differentiation of preantral follicles are of considerable interest, since their elucidation is a prerequisite to use of the primordial pool to enhance reproductive efficiency in domestic animals, humans, and endangered species. This review is an attempt to summarize the approaches that have been taken to further this goal and the results thus far of these efforts. Preantral follicular development can be divided into three stages: activation of primordial follicles, the primary to secondary follicle transition, and the development of secondary follicles to the periantral stage. The activation of primordial follicles in vitro has been achieved thus far in rodents, cattle, and primates, where it occurs spontaneously without the addition of growth factors or hormones. The ovaries of rodents are small enough to be cultured intact and, in that experimental situation, some follicles activate, while many remain in the resting pool, and the addition of specific factors can increase or decrease the number of follicles that leave the resting pool in vitro. In contrast, follicular activation in cattle and primates has been studied by culturing small pieces of the ovarian cortex, rich in primordial follicles, and the great majority of the primordial follicles activate in that situation, suggesting the importance of inhibitory factors to the normal, gradual exit of follicles from the resting pool. In cultured rodent ovaries, follicles appear to pass easily and spontaneously from the primary to the secondary stage, whereas few of the activated follicles in cultured cortical pieces from cattle or primates progress from the primary to the secondary stage. Understanding the requirements for the primary to secondary transition is critical for growing follicles activated in vitro to the late preantral and antral stages. In contrast, the requirements for the continued growth of larger preantral follicles, which can be isolated for in vitro studies, have been extensively explored in rodents and to a lesser extent in domestic species. A number of hormones and factors have been implicated and will be discussed. Taken together, the results highlight the need for a better understanding of the earliest stages of follicular development in domestic ruminants, particularly follicle activation and the primary to secondary follicle transition.     

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime’s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.     

Live offspring by in vitro fertilization of oocytes from cryopreserved primordial mouse follicles after sequential in vivo transplantation and in vitro maturation

The objectives of the present study were to achieve 1) oocyte maturation, 2) oocyte competence of fertilization, and 3) oocyte competence of embryogenesis with oocytes from primordial follicles obtained from cryopreserved newborn mouse ovaries by using a two-step method. In the first step, frozen-thawed newborn mouse ovaries were transplanted under the kidney capsule of recipients for the initiation of growth from the primordial follicle stage on. In the second step, growing preantral follicles in the ovarian grafts were recovered and cultured. The results demonstrated that primordial follicles were able to be recruited to preantral follicles during the period of transplantation, and preantral follicles could be mechanically isolated from ovarian grafts. Under the present in vitro culture conditions, 85.8% of the isolated follicles (n = 332) from ovarian grafts survived the 12-day in vitro culture process, 84.9% of the recovered oocytes (n = 285) were germinal vesicle breakdown (GVBD)-competent, and 76% of the oocytes that underwent GVBD (n = 242) developed to the metaphase II (MII) stage. In the in vitro fertilization experiments, 75.4% of 142 inseminated MII oocytes underwent fertilization and cleavage to the 2-cell stage. Subsequently, 79.7% of the 2-cell-stage embryos (n = 69) progressed to the late morula-early blastocyst stage. Transfer of late morula-early blastocyst embryos resulted in the production of live offspring. From our experiments, it may be concluded that in vivo maturation by grafting followed by in vitro maturation of frozen-thawed primordial follicles can restore fertility in mice. This model could be useful for a similar application in the human.     

Comparison of three in vitro culture systems for maturation of early preantral mouse ovarian follicles

The aim of this study was to compare three different culture systems for in vitro follicular growth and oocyte maturation in ovarian follicles of mice in order to assess the technique with the optimal growth and improved rate of meiotic maturation. The three systems tested were culture under oil, on a hydrophobic membrane and on agar respectively. Early preantral follicles were cultured for 12 days in alpha-MEM GlutaMAX medium. Follicular growth, oocyte meiotic maturation, oocyte extrusion, atresia and estradiol production were analysed. Follicular development showed two phases in the three systems, with slow growth before day 5 and subsequent acceleration. The percentage of follicles transferred into oocyte maturation medium was significantly higher after culture under oil. The proportion of oocytes that achieved nuclear maturation (metaphase II) was higher when follicles were cultured under oil or on a hydrophobic membrane than on agar. Our results support the use of culture under oil for in vitro follicular growth from the early preantral stage in order to obtain metaphase II oocytes. Fertilization ability of these oocytes and the capacity to obtain healthy mice in a reproducible manner warrants further investigation.     

Comparison of preimplantation developmental competence after mouse oocyte growth and development in vitro and in vivo

Culture systems for oocytes are essential for the experimental analysis of the basic mechanisms of oocyte development and, moreover, they will eventually find wide application in agriculture, the clinic, and wildlife preservation. Here, progress in mouse oocyte growth and development in vitro using oocyte-granulosa cell complexes from preantral follicles is reviewed. Oocyte-granulosa cell complexes were isolated from preantral (secondary) follicles of 12 day old mice, grown in vitro for 10 days, then matured and fertilized in vitro. The developmental competence of these oocytes was compared with oocytes grown in vivo and isolated from 22 day old mice, then matured and fertilized in vitro. In vitro-grown oocytes did not achieve the same size as their in vivo-grown counterparts. However, when oocytes were grown in medium containing fetal bovine serum, their preimplantation developmental competence was equivalent to that of in vivo-grown oocytes. Surprisingly, more blastocysts per animal were produced when oocytes were grown in vitro than in vivo. There was no correlation between oocyte size and either preimplantation developmental competence or number of cells per blastocyst. Oocytes grown in serum-free medium did not achieve the same developmental competence as oocytes grown in medium supplemented with serum. Lastly, the health status as an adult of the only animal born after complete oocyte development in vitro is described and discussed.     

Growth and antrum formation of bovine preantral follicles in long-term culture in vitro

Culture of preantral follicles has important biotechnological implications through its potential to produce large quantities of oocytes for embryo production and transfer. A long-term culture system for bovine preantral follicles is described. Bovine preantral follicles (166 +/- 2.15 micrometer), surrounded by theca cells, were isolated from ovarian cortical slices. Follicles were cultured under conditions known to maintain granulosa cell viability in vitro. The effects of epidermal growth factor (EGF), insulin-like growth factor (IGF)-I, FSH, and coculture with bovine granulosa cells on preantral follicle growth were analyzed. Follicle and oocyte diameter increased significantly (P < 0.05) with time in culture. FSH, IGF-I, and EGF stimulated (P < 0.05) follicle growth rate but had no effect on oocyte growth. Coculture with granulosa cells inhibited FSH/IGF-I-stimulated growth. Most follicles maintained their morphology throughout culture, with the presence of a thecal layer and basement membrane surrounding the granulosa cells. Antrum formation, confirmed by confocal microscopy, occurred between Days 10 and 28 of culture. The probability of follicles reaching antrum development was 0.19 for control follicles. The addition of growth factors or FSH increased (P < 0.05) the probability of antrum development to 0.55. Follicular growth appeared to be halted by slower growth of the basement membrane, as growing follicles occasionally burst the basement membrane, extruding their granulosa cells. In conclusion, a preantral follicle culture system in which follicle morphology can be maintained for up to 28 days has been developed. In this system, FSH, EGF, and IGF-I stimulated follicle growth and enhanced antrum formation. This culture system may provide a valuable approach for studying the regulation of early follicular development and for production of oocytes for nuclear/embryo transfer, but further work is required.     

Understanding follicle growth in vitro: Are we getting closer to obtaining mature oocytes from in vitro‐grown follicles in human?

Obtaining and fertilizing mature oocytes from immature follicles that were grown outside the body has conceptually attracted scientists for centuries, with initial attempts first documented in the 19th century. Significant progress has been made since then, due in part to a better understanding of folliculogenesis and improved techniques of in vitro follicle growth. Indeed, in vitro growth is now considered a reasonable approach to preserve or restore fertility when immature follicles and their oocytes need to be grown and matured outside the body. Certain patients would benefit from in vitro follicle growth, particularly those who carry a risk of cancer re‐seeding after grafting of frozen‐thawed ovarian tissue or who are at the risk of premature ovarian failure due to several intrinsic ovarian defects and genetic mutations that lead to accelerated follicle atresia and early exhaustion of the ovarian reserve. This review provides an update on the current status of in vitro growth of preantral human follicles, from initial efforts to the most recent achievements.     

The stage-dependent inhibitory effect of porcine follicular cells on the development of preantral follicles

The objective of this study was to examine the effects of follicular cells on the in vitro development of porcine preantral follicles. In Experiment 1, one preantral follicle alone (Trt 1) was cocultured with a follicle of the same size with oocytes (Trt 2) or without oocytes (Trt 3). Preantral follicles cultured alone in vitro for 12 days had greater follicle diameters (1017 +/- 96 microm versus 706 +/- 69 or 793 +/- 72 microm, P < 0.05), growth rates (201 +/- 0.3 versus 103 +/- 0.2 or 128 +/- 0.2, P < 0.05) and oocyte survival rates (73% versus 48, or 25%, P < 0.05) than other groups. The inhibitory effects of follicle cells on the growth of preantral follicles and oocyte survival rates were not enhanced by the addition of oocytectomized preantral follicles (Experiment 2). Follicles were cocultured with different sources of follicular cells in other experiments. Coculture with cumulus cells enhanced oocyte survival compared to the control (without coculture) and mural follicular cell groups (Experiment 3). The growth and survival rates of oocytes collected from the group of follicles cocultured with cumulus cells from large antral follicles (>3 mm) were greater (P < 0.05) than those from small antral follicles (<3 mm), or than the control group (without cumulus cells, experiment 4). No significant differences in the follicular diameters (674 +/- 30 microm versus 638 +/- 33 and 655 +/- 28 microm) and growth rate (105% versus 94 and 105%) were observed among the preantral follicles of the different treatments (P > 0.05). Taken together, coculture with the cells from large antral follicles (>3 mm) exerted a significant positive effect on oocyte survival. The growth and oocyte survival of preantral follicle cocultured with the same size of follicles (with or without oocyte) were inhibited. Growth and survival rates of preantral follicles and oocytes are improved by coculturing them with the cumulus cells derived from larger antral follicles.     

Production of buffalo embryos using oocytes from in vitro grown preantral follicles

The present study examines the use of buffalo preantral follicles as a source of oocytes for in vitro embryo production. Preantral follicles were isolated from abattoir-derived buffalo ovaries and were grown for 100 days in five different culture systems: (1) minimum essential medium (MEM); (2) coconut water; (3) MEM + ovarian mesenchymal cell (OMC) co-culture; (4) MEM + granulosa cell (GC) co-culture; or (5) MEM + cumulus cell (CC) co-culture. Low growth rates for the preantral follicles were observed when follicles were cultured in MEM or coconut water medium. Moderate growth rates were seen for OMC and GC co-cultures, and high rates of growth were observed when follicles were grown in CC co-culture. The survival of preantral follicles was low in the MEM culture (<25%), but was over 75% in the other culture systems. Oocytes were not recovered from the MEM group, while an oocyte recovery rate of 80-100% was observed when the follicles were cultured with coconut water/somatic cells. Transferable embryos could be produced only with the oocytes obtained from preantral follicles grown in the OMC and CC co-culture systems. This study demonstrates, for the first time, that it is possible to produce buffalo embryos by in vitro fertilization of oocytes derived from in vitro grown preantral follicles.     

Xenotransplantation in immunodeficient mice to study ovarian follicular development in domestic animals

Nowadays, in vitro study of follicular dynamics of primordial and primary follicular stages is limited because in vitro culture systems for these follicles are lacking, both in domestic animal species and in human. Therefore, additional insights might be generated by grafting ovarian tissue into immunodeficient mice to study activation and maturation of early follicular stages. A considerable amount of data has already been gathered in laboratory animals and through clinical application of human assisted reproduction technologies where live births were reported recently after the use of (cryopreserved) ovarian grafts. However, given that human preantral follicles are difficult to obtain and that there are many similarities between the bovine and human species with regard to ovarian physiology, the bovine model offers exciting additional prospects and is therefore discussed in more detail. This review will focus on recent developments related to preantral follicle and (repeated) ovarian tissue retrieval and xenotransplantation of (bovine) ovarian tissue strips to immunodeficient mice as a model to study preantral follicular dynamics. Different grafting strategies will be discussed as well as the consequences of this procedure on the viability and dynamic behavior of the grafted tissue and follicles.