Animal Models in Biotechnologies for Male Fertility Preservation

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1		Manuel Garcia-Herreros	--	5304	2023-12-27 17:28:58	\|
2	update references	Rita Xu	Meta information modification	5304	2023-12-28 03:23:41	\| \|
3	update layout	Rita Xu	Meta information modification	5304	2023-12-28 03:25:08	\|

This entry is adapted from the peer-reviewed paper 10.3390/life14010017

To explore advanced reproductive technologies for male fertility preservation, underscoring the essential role that animal models have played in shaping these techniques through historical contexts and into modern applications. Rising infertility concerns have become more prevalent in human populations recently. The surge in male fertility issues has prompted advanced reproductive technologies, with animal models playing a pivotal role in their evolution.

animal models male infertility spermatogenesis spermatogonial stem cells (SSCs)

1. Introduction

Over the past few decades, the domain of reproductive biomedicine has been at the forefront of profound technological advancements, highlighting the escalating imperative to preserve male fertility. As the global incidence of male infertility continues to rise due to many factors—from lifestyle changes and environmental factors to genetic predispositions—there is an augmented interest in innovative methods to mitigate these challenges. Historically, most of these advancements have been made possible through the extensive use of animal models, allowing researchers to explore, test, and refine techniques in a controlled setting before their application to human subjects.

1.1. Overview of Male Fertility Issues

Human male fertility has witnessed a concerning decline over recent decades, with various studies indicating a notable decrease in sperm counts, motility, and overall male reproductive health. This reduction in male fertility is multifaceted, encompassing a wide array of physiological and environmental causes. Physiological challenges include genetic abnormalities, hormonal imbalances, and various health conditions like varicocele or infections affecting the reproductive tract. At the genetic level, anomalies like Y-chromosome microdeletions or mutations in specific genes can lead to sperm production issues. Hormonal imbalances, on the other hand, can affect the entire gametogenesis process, leading to reduced sperm production or compromised sperm health.

Beyond these physiological challenges, environmental and lifestyle factors have significantly contributed to the declining male fertility trend. Exposures to endocrine-disrupting chemicals (EDCs), commonly found in plastics, pesticides, and industrial chemicals, can interfere with hormonal pathways crucial for sperm production and maturation. Additionally, lifestyle elements such as poor diet, smoking, excessive alcohol consumption, obesity, and even psychological stress have been associated with adverse effects on sperm quality and overall male reproductive capability. As the global community grapples with these diverse challenges, the quest for effective and innovative fertility preservation techniques becomes even more paramount.

Classification of Human Infertility Conditions

Causes of male factor infertility can be divided into pre-testicular, testicular, and post-testicular factors. Other specific groups of conditions include immunological, environmental, and idiopathic. Pre-testicular factors refer to conditions or influences outside of the testicles that can impact sperm production or function. These can include the following: testicular factors, which pertain to conditions or issues directly related to the testes that affect their ability to produce healthy sperm; and post-testicular factors, which refer to conditions or obstructions that affect the transport or delivery of sperm post-production in the testes. These can include issues with parts of the male reproductive tract that transport, store, or protect sperm, such as the efferent ducts, epididymis, vas deferens, male glands, and urethra.

Immunological factors usually include the production of auto-anti-sperm Antibodies. Some men produce antibodies that attack their sperm, harming their fertility ^[1].

1.2. Animal Models and Male Infertility Overview

Animal models have been pivotal in understanding the fundamental aspects of male reproduction and developing and refining novel interventions for fertility preservation.

Techniques such as intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) were initially honed in animals. With their fast breeding and well-understood reproductive biology, rodents have been particularly instrumental, while larger animals like rabbits and non-human primates offer a closer physiological match to humans. These models are not only crucial for genetic studies, including gene knockout and overexpression techniques, but also for examining the impact of environmental and lifestyle factors on fertility. Recent use of animal models extends to assessing the safety and efficacy of new technologies like mitochondrial replacement therapy and gene editing. Beyond research, they are invaluable for training in various reproductive procedures. Despite their critical role, it is essential to acknowledge their limitations, as they do not fully replicate human biology, necessitating careful interpretation of results derived from these models.

2. Advances in Male Fertility Preservation Techniques

2.1. Animal Studies and Implications for Human Fertility

Animal models have been instrumental in advancing the field of artificial gamete generation, offering invaluable insights with implications for human fertility treatments. While mice models have proven pivotal in establishing protocols to study male infertility, there remain significant challenges when translating these accomplishments into human applications. One of the predominant challenges arises from ethical and regulatory considerations. For instance, the use of human embryonic stem cells in research is often enveloped in ethical debates and stringent regulations ^[2].

Biologically, humans and mice present notable differences. Fine-tuning techniques for mice may not directly apply to humans due to variations in germ cell developmental timelines, among other factors. Additionally, technical challenges persist, and many developments remain experimental to date.

2.2. Artificial Gametes

Artificial gametes, often referred to as in vitro-derived gametes or synthetic gametes, represent a cutting-edge frontier in reproductive biology. These are sex cells—sperm or eggs—generated outside the human body, typically derived from germline stem cells. Once germ line cell types are obtained, they can be converted from the least differentiated state to the most differentiated. Their potential application in treating infertility and broadening reproductive options offers a transformative approach to reproductive medicine.

2.2.1. Deriving Germ Line Cells from Pluripotent Stem Cells: Embryonic and Induced Pluripotent Stem Cells

In the intricate landscape of cell biology, pluripotent stem cells stand out for their exceptional ability to differentiate into virtually any cell type in the human body, including germ line cells. In creating artificial gametes, the potential to derive germ line cells from pluripotent stem cells represents a crucial advancement. It provides a practical approach to tackling reproductive challenges like infertility and contributes to understanding the basic mechanisms of human reproduction. Embryonic stem cells (ESCs) are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early stage embryo. Due to their pluripotency, ESCs can differentiate into any of the three germ layers: endoderm, mesoderm, or ectoderm. This ability to transform into diverse specialized cell types makes them invaluable for research, therapeutic applications, and understanding early human development. The first establishment of mouse embryonic stem cells (ESCs) was achieved by two researchers independently: Martin Evans and Matthew Kaufman at the University of Cambridge in 1981 and Gail R. Martin at the University of California, San Francisco, also in 1981 ^[3]^[4].

Induced pluripotent stem (iPS) cells are a type of pluripotent stem cell that can be generated directly from adult cells, effectively bypassing the need for embryos. Professor Shinya Yamanaka and his team at Kyoto University pioneered the concept of iPS cells in 2006. They successfully reprogrammed mature mouse fibroblasts into pluripotent stem cells by introducing a combination of four specific genes, now famously known as the “Yamanaka factors”: Oct4, Sox2, Klf4, and c-Myc. These factors act as molecular switches that reset the adult cells into a pluripotent state, mirroring the properties of embryonic stem cells ^[5].

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are promising for deriving germ line cells due to their pluripotent properties. Studies have shown that under specific culture conditions, both ESCs and iPSCs can differentiate, for instance, into primordial germ cell-like cells (PGCLCs) ^[6]^[7]^[8]. These cells can further develop into more mature germ cells, including spermatogonia, spermatocytes, and even spermatids in vitro. The induction of PGCLCs from pluripotent stem cells involves mimicking the signaling events that occur during early embryogenesis, typically involving the modulation of key signaling pathways such as BMP ^[8] and the expression of key germ cell genes. This ability to generate germ cells from pluripotent stem cells offers significant potential for studying the mechanisms of germ cell development and could potentially be used for therapeutic applications in infertility treatments.

2.2.2. Mouse Models for Artificial Gametes Generation

Both Toyooka’s and Geijsen’s teams independently succeeded in generating primordial germ cells (PGCs) from embryonic stem (ES) cells. Toyooka’s group successfully achieved in vitro production of functional germ cells from embryonic stem (ES) cells ^[8]. They utilized knock-in ES cells where GFP or lacZ was expressed from the endogenous mouse vasa homolog (Mvh), a marker specifically expressed in differentiating germ cells. The approach used allowed them to visualize germ cell production during in vitro differentiation. The emergence of MVH-positive germ cells was contingent on embryoid body formation and was significantly enhanced by bone morphogenic protein 4-producing cells. When these ES-derived MVH-positive cells were transplanted into reconstituted testicular tubules, they participated in spermatogenesis, confirming that the ES cells could produce functional germ cells in a laboratory setting. They progressed further by deriving gonocytes from the PGCs and, subsequently, spermatogonial stem cells (SSCs) from these gonocytes.

On the other hand, Geijsen’s group produced spermatid-like cells, termed haploid-like (HL) cells, from embryonic stem cells ^[6]. They utilized embryonic stem cells in embryoid body systems. Since embryoid bodies sustain blood development, the researchers hypothesized that these structures might also support the formation of primordial germ cells, the founder population of gametes in mice. By isolating primordial germ cells from embryoid bodies, they were able to derive continuously growing lines of embryonic germ cells. These germ cells displayed erasure of methylation markers (imprints) characteristic of the germ lineage. They also demonstrated that embryoid bodies support the maturation of primordial germ cells into haploid-like male gametes. When these gametes were injected into oocytes, they restored the somatic diploid chromosome complement and developed into blastocysts, though live offspring were not achieved.

In a pivotal 2006 study, Nayernia et al. developed both SSCs and sperm cells from mouse ESCs ^[9]. By using ICSI with these artificially produced sperm, they generated viable embryos. Normal offspring were produced when these embryos were transferred to surrogate mothers, marking a significant milestone in the field. Further advancements were observed with induced pluripotent stem (iPS) cells. Hayashi’s team, 2011, produced epiblast cells from which they derived PGCs ^[10]. By 2012, Zhu’s team had generated both SSCs and HL cells from iPS cells ^[11]. Imamura’s study stood out as they first created iPS cells from hepatocytes and then successfully derived PGCs from these cells ^[7].

Ishikura et al. (2021) pioneered a methodology to systematically derive the entire spectrum of male germ cell types from pluripotent stem cells ^[12]. In their study, day 4 cultured mouse primordial germ cell-like cells (mPGCLCs) derived from mESCs were employed as starting materials to differentiate them into spermatogonium-like cells. These cells were then developed into germline stem cell-like cells (GSCLCs) that exhibited robust spermatogenesis both in vivo and in vitro. The study highlighted the significance of genome-wide DNA demethylation for proper spermatogonia differentiation. GSCLCs derived under these conditions showed a transcriptome and DNA methylome closely resembling genuine germline stem cells, though some differences persisted. These differences may impact the function of spermatogonia, but the derived GSCLCs still demonstrated efficient spermatogenesis and produced viable offspring through techniques like ICSI/ROSI. The study emphasizes that while the whole process of male germ-cell development has been reconstituted in vitro with the assistance of embryonic/neonatal testicular somatic cells, future challenges involve inducing specific stages of germ-cell development under defined conditions without relying on testicular somatic cells. The methods and approaches pioneered in this research offer insights into male germ-cell development, its challenges, and potential therapeutic solutions.

iPSCs vary in their states across species. Specifically, mouse iPSCs are predominantly in the naïve state, whereas human iPSCs tend to be in the primed state. Similarly, mouse ESCs are generally considered naïve in contrast to human ESCs, which are in the primed state ^[13]. These distinct states demand growth factors for self-renewal and can be transitioned between specific culture environments. This distinction is essential for understanding the developmental potential of these cells to derive human germ line cells.

2.2.3. Artificial Gametes Generation in Humans

Several research groups have successfully produced primordial germ cells (PGCs) from human embryonic stem cells (ESCs), including teams led by Kee (2009), Bucay (2009), Tilgner (2008 and 2010), and Aflatoonian (2009) ^[14]^[15]^[16]^[17]. Notably, Aflatoonian (2009) and Kee (2009) advanced this work by achieving the production of haploid-like (HL) human cells from ESCs. Regarding induced pluripotent stem (iPS) cells, Eguizabal’s groundbreaking 2010 study generated PGCs, marking an important milestone in the field ^[18]. Panula et al. (2011) mirrored this achievement and took it a step further by producing HL human cells from iPS cells ^[19]. To date, no research group has been able to create human embryos or offspring using artificial male gametes. As researchers discover more about deriving germ cells from pluripotent cells, certain human embryonic stem cell lines, notably H1, H7, and H9, have emerged as instrumental tools ^[20]. H1 and H9, in particular, have been highlighted as commonly requested by the National Stem Cell Bank ^[20].

In the seminal year of 2004, Clark and colleagues paved the way with their observations on embryoid body (EB) differentiation, revealing that spontaneous differentiation of human embryonic stem cells (hESCs) was capable of giving rise to putative germ cells expressing a myriad of genes, including DAZL, DPPA3, DDX4, and SYCP3 ^[21].

In 2015, it was learned that the pre-inducing hPSCs to specific states, such as a distinct pluripotent state or a mesoderm-like state, before venturing into direct germ-cell differentiation was beneficial for generating germ cell line cells ^[22]^[23]. Deriving germ cells from embryoid bodies is the emerging strategy, as demonstrated by both these research groups. At the same time, Irie et al. utilized mouse embryonic fibroblasts (MEFs) followed by a priming culture on vitronectin/gelatin ^[23], and Sasaki et al. initiated their cultures on laminin, they primed on plasma fibronectin ^[22]. In their research, Sasaki et al. pinpointed specific culture conditions and signaling pathways conducive to the efficient induction of human germ cell-like cells from pluripotent stem cells (hPSCs). The cells they derived displayed characteristics that aligned with those of early germ cells. Irie et al. delved into the pivotal role of the SOX17 transcription factor in determining the fate of human primordial germ cells (PGCs). Their findings highlighted SOX17’s essentiality in guiding human PGC development, a marked contrast to the mouse model where BLIMP1 assumes a dominant function. This study shed light on the intricate molecular mechanisms orchestrating human PGC specification.

More recently, a meticulous comparison of the gene expression profile across five distinct human embryonic stem cell lines cultivated on varied matrices was conducted. This study revealed that after several passages on laminin 521, a more homogeneous expression pattern of key pluripotency markers, including POU5F1, NANOG, SOX2, and GDF3, was discernible ^[20].

To date, the methodology for assessing the functionality of human germ cells derived from hPSCs has primarily revolved around protein- and gene-expression analyses. Key protein markers, including DDX4, POU5F1, SYCP3, DAZL, cKIT, PRDM1, SSEA1, DPPA3, and acrosin, have been employed to identify various stages and types of germ cell differentiation ^[20]. However, unlike in animals where functionality is confirmed by observing changing DNA contents during meiosis and the production of viable offspring using in vitro generated gametes, a definitive gold standard methodology for humans remains elusive due to ethical constraints.

Single-cell RNA sequencing (RNA-seq) has advanced the comprehension of germ-cell development, shedding light on the maturation trajectory from prenatal stages to adulthood. Irie et al. and Sasaki et al. have employed RNA-seq to highlight the critical roles of genes like SOX17 and to compare hPGCLCs with other germ cells ^[22]^[23]. This technology has unveiled the intricacies of adult germ cells, including identifying multiple distinct populations ^[24]. Furthermore, it has emphasized the significance of the somatic environment in germ cell development, with recent studies underscoring the potential of three-dimensional culture conditions in aiding differentiation ^[25]. Through RNA-seq, researchers can better refine differentiation protocols and compare in vitro germ cells to their in vivo counterparts ^[20].

While these groundbreaking studies have advanced the understanding of human germ cell differentiation, further research is imperative to bridge the gap between these findings and the successful generation of fully functional human artificial gametes.

2.2.4. Other Animal Models for Artificial Gametes Generation

Rat. The rat is a crucial biomedical research model, with its pluripotent stem cells offering unprecedented insights into reproductive medicine. Recent studies have highlighted the potential of rat-derived pluripotent stem cells in germline transmission and the production of viable offspring. Hamanaka’s pivotal study underscored the capability of riPSCs to contribute to germline transmission. Through the reprogramming of rat somatic cells using three key factors—Oct3/4, Klf4, and Sox2—riPSCs were derived, showing competence in contributing to both intraspecific rat and interspecific mouse-rat chimeras ^[26]. The next logical step was the generation of Functional germ cells from Pluripotent Stem Cells in Rats. Oikawa’s research aimed to derive functional primordial germ cell-like cells (PGCLCs) from rat pluripotent stem cells. Their findings demonstrated the successful induction of PGCLCs capable of producing functional spermatids, which subsequently sire viable offspring ^[27].

Iwatzuki et al. made significant strides in deriving and propagating post-implantation epiblast-derived pluripotent stem cells (rEpiSCs). Optimizing culture conditions, they revealed rEpiSCs’ potential to be reset to a naive pluripotent state using exogenous Klf4. Crucially, these rEpiSCs demonstrated competency in generating primordial germ cell-like cells, leading to functional gametogenesis and the birth of viable progeny Link to source.

Ming-Gui Jiang and colleagues presented a groundbreaking methodology optimizing induction media, notably with knock-out serum replacement and vitamin C. Their approach facilitated the efficient derivation of riPSCs from Dark Agouti rat fibroblasts and Sertoli cells. These riPSCs, exhibiting stable undifferentiated states over 30 passages, differentiated into various cell types, including germ cells. A notable achievement was the production of transgenic riPSCs using the PiggyBac transposon, setting the stage for transgenic rat creation via germ line transmission. Their success in obtaining transgenic offspring using the derived gametes positions riPSCs as a valuable tool for rat genetics and genomics, emphasizing their relevance in artificial gamete derivation ^[28].

In summary, these groundbreaking advances in rats echo similar accomplishments observed in mice, highlighting the potential of these phylogenetically related animal models in pioneering the fields of reproductive biology and genetic engineering.

Rabbits. In rabbits, primordial germ cell (PGC) specification happens at the posterior epiblast at the beginning of gastrulation, similar to the development in bilaminar discs observed in humans and most mammals, contrasting with rodent development as egg cylinders ^[29]. From newly derived rabbit pluripotent stem cells, rbPGC-like cells can be robustly and rapidly induced in vitro using WNT and BMP as morphogens, and therefore, SOX17 identified as the pivotal regulator of rbPGC fate, consistent with its role in several non-rodent mammals ^[29]. The study suggests that the development of bilaminar discs is a key factor determining PGC regulators, independent of the diverse development of extraembryonic tissues.

Pluripotent stem cell lines have been derived from rabbits ^[30]. These cell lines express stem cell-associated markers and maintain apparent pluripotency during multiple passages in vitro. However, their complete in vivo pluripotency has yet to be convincingly demonstrated. The difficulty in achieving fully pluripotent stem cell lines in rabbits, as compared to mice, is due mainly to suboptimal rabbit markers for embryonic stem cells (ESCs), which are not always specific to the pluripotent inner cell mass. Besides, efficient somatic cell reprogramming requires rabbit-specific pluripotency genes, which are currently challenging to identify and utilize. While germ line cell types have not yet been derived from rabbits, this animal model holds promise for artificial gamete generation to address human infertility issues. The rabbit model offers advantages, such as its physiological similarities to humans and a shorter reproductive cycle, making it a potential candidate for reproductive research.

2.3. Spermatogonial Stem Cell-Based Therapies

Spermatogonial stem cells (SSCs) are critical components of male fertility, maintaining a constant pool of cells in the testis and facilitating sperm production through spermatogenesis. Their unique properties lend them to promising animal reproduction and regenerative medicine applications. These applications include gene targeting, inducing pluripotency, and potentially restoring fertility. Techniques such as SSC transplantation and testis tissue xenografting have been developed, though these remain technically challenging in large animals and humans. Advancements in SSC culture methods have further expanded their potential use. In addition, the successful demonstration of in vitro spermatogenesis in mice offers exciting potential for addressing reproductive issues in the agricultural sector and human fertility treatments.

2.3.1. SSCs Transplantation

The development and application of spermatogonial stem cell (SSC) transplantation can be traced back to the late 20th century. In 1994, a foundational study by Brinster and Zimmerman demonstrated that germ cells could colonize mouse testes and initiate spermatogenesis, setting the stage for subsequent investigations into spermatogonial stem cell (SSC) transplantation ^[31]. Two years later, in 1996, Brinster’s lab conducted a seminal experiment where they successfully transplanted frozen-thawed spermatogonial stem cells into the seminiferous tubules of recipient mice ^[32]. The ability of these cells to recolonize, differentiate, and commence spermatogenesis confirmed the stem cell properties of spermatogonial stem cells.

The early 2000s saw the technique expanded to larger animal models. Between 2002 and 2003, successful SSC transplantations were reported in goats, boars, and cattle ^[33]^[34]^[35]. These results suggested the broader applicability of the technique beyond small mammals. By 2013, another significant advancement occurred when SSC transplantation was successfully used to restore monkey fertility ^[36], highlighting the potential for its application in primates.

From 2012 onwards, research has been geared towards refining the SSC transplantation methodology. Focus areas include improving colonization and spermatogenesis efficiency and evaluating the associated risks. Additionally, there’s an ongoing exploration of the technique’s clinical application, particularly for fertility preservation in prepubertal boys undergoing treatments such as chemotherapy.

The development of SSC transplantation clearly illustrates the advances that have been made in this field over the past few decades. Still, it highlights the challenges before this procedure can be widely applied in clinical practice.

Spermatogonial stem cell (SSC) transplantation is a procedure that involves isolating SSCs from a donor testis and transplanting them into the testis of a recipient. The transplanted SSCs then colonize the seminiferous tubules and initiate spermatogenesis, thereby producing sperm carrying the genetic material of the donor. The potential of SSC transplantation for overcoming fertility problems is significant. SSC transplantation holds substantial possibilities for addressing fertility challenges based on several key applications. One primary application is in the realm of oncology. Men diagnosed with cancer often undergo treatments, such as chemotherapy or radiation therapy, which carry the risk of germ cell damage leading to infertility ^[37]. SSC transplantation offers a potential solution. Before commencing these treatments, SSCs could be harvested and cryopreserved. Post-treatment, these cells could be transplanted back, aiming to restore fertility.

Genetic anomalies are among the causes of male infertility. SSC transplantation introduces a therapeutic avenue whereby SSCs from an infertile patient are genetically modified ex vivo to rectify the inherent genetic defect. Once corrected, these cells could be reintroduced into the patient’s testes to potentially fix the infertility issue.

From a research perspective, SSC transplantation offers an unparalleled tool ^[38]. It enables the study of male germ cell development and spermatogenesis mechanisms. Understanding these processes in depth could pave the way for innovative treatments addressing male infertility. It’s worth noting that while these potential applications are promising, SSC transplantation is still mainly in the experimental stage. There are technical challenges to be overcome, such as the low efficiency of colonization of the transplanted SSCs and potential risks, such as the risk of transmission of diseases or abnormal cells. Therefore, more research is needed before this technique can be widely used in clinical practice.

2.3.2. Animal Models Involving SSCs

Mouse. Spermatogonial stem cell (SSC) transplantation in mice involves the collection of SSCs from a donor mouse’s testes and their subsequent introduction into the seminiferous tubules of an infertile recipient mouse. To prepare the recipient, its native germ cells are typically eradicated using treatments such as busulfan or irradiation to create a niche for the incoming SSCs. Once the donor SSCs are isolated, they are injected into the rete testis of the recipient, from where they migrate to the seminiferous tubules. These transplanted SSCs then colonize the recipient’s testes, differentiate, and initiate the process of spermatogenesis, allowing the previously infertile mouse to produce sperm derived from the donor’s genetic material. Brinster et al. 1994 found that cells derived from the testis and transplanted into an infertile mouse testis can establish residence in seminiferous tubules and begin the process of spermatogenesis in over 18–37% of the recipient mice ^[31].

Farm animals. Spermatogonial stem cell (SSC) transplantation has also been explored extensively in farm animals as they hold great promise as animal models for infertility and in various aspects of livestock management and genetic improvement.

Approaches with primates. In 2012, researchers reported successfully using SSC transplantation to restore fertility in rhesus macaque monkeys ^[39]. Autologus spermatogonial stem cells were transplanted into monkeys rendered infertile due to chemotherapy. Following transplantation, embryos with donor paternal origin were produced. This study was significant because it demonstrated the potential of SSC transplantation in primates, bringing the technique closer to potential applications in humans.

2.4. Spermatogenesis In Vitro

Animal models have dramatically facilitated understanding the complexities of human male infertility, particularly the transformation of spermatogonial stem cells into mature spermatozoa. These models have been instrumental in in vitro spermatogenesis, a promising area with implications for both understanding and addressing human male infertility.

In a 2011 study by Sato et al., researchers successfully replicated the process of spermatogenesis in vitro using neonatal mouse testes. They produced viable sperm that resulted in healthy offspring and demonstrated the potential of cryopreserving these tissues for future applications, paving the way for further advancements in reproductive biology and medicine ^[40]. In the study on in vitro spermatogenesis, several key elements were pivotal in successfully reproducing this intricate biological process. Firstly, the researchers harnessed the potential of neonatal mouse testes, rich in gonocytes or primitive spermatogonia. These early stage germ cells provided an optimal starting point for initiating and sustaining spermatogenesis in an artificial environment. Secondly, choosing a serum-free culture media eliminated any inconsistencies that serum components might introduce, ensuring a controlled and supportive environment for the germ cells to thrive. Furthermore, the strategic positioning of the testes tissue fragments at the gas-liquid interphase was instrumental. This placement ensured optimal oxygenation, a critical cell differentiation and proliferation factor. Validating the efficacy of their methods, the researchers successfully used the in vitro-derived spermatids and sperm to produce healthy and reproductively competent offspring via ICSI. This demonstrated the functional quality of the produced germ cells and marked a significant achievement in the realm of reproductive biology. In humans, the process has been achieved up to the stage of elongated spermatids ^[41]^[42], but offspring have not yet been obtained. Meanwhile, in the domain of farm animals, the bovine model stands as a prominent example of advancements in this field.

The ideal in vitro spermatogenesis culture system would likely be a three-dimensional (3D) model that closely mimics the testicular microenvironment. This system should permit cells to interact within a matrix and with each other, thereby replicating natural tissue architecture. The culture method should incorporate both testicular somatic cells and germ cells, allowing for complete spermatogenesis. The system should not only support the development of spermatogonial cells, but also facilitate the progression of meiosis and ensure the functionality of haploid cells. The balance and composition of somatic cell populations would be crucial, and the incorporation of spermatogonia into reconstructed tubular structures should be efficient. Additionally, the ideal culture system should account for the role of hormones and other key molecules that promote spermatogenesis, such as those identified by Sanjo et al. in 2018 and 2020. Ensuring that these factors are present in the three-dimensional culture environment would likely enhance the process of spermatogenesis, leading to the production of functional spermatids.

2.5. Xenotransplants of Testicular Tissue

Testicular tissue xenografting is a technique wherein pieces of testicular tissue are implanted into immunocompromised mice, usually in the back subcutaneous tissue, facilitating the explant to grow and subsequently produce sperm. This groundbreaking method was introduced in 2002 when researchers successfully transplanted testicular tissue from immature pigs and goats into nude mice, evidencing its potential for fertility preservation ^[43]. The grafted tissue establishes a functional circulatory connection with the host mouse ^[44]. After forming this connection, a functional feedback loop is created between the mouse’s pituitary and the endocrine cells in the graft, leading to the growth of the xenografts and sperm production over time ^[45], sperm that even though did not undergo maturation in the epididymis, is still potent for fertilization when employed in intracytoplasmic sperm injection (ICSI) ^[45]. The technique’s efficacy has been demonstrated across various species, with sperm from the xenografts being utilized to initiate fertilization. In fact, the production of viable offspring has been reported in rabbits and pigs using this method ^[46]^[47]^[48] and even humans. In a groundbreaking study, cryopreserved prepubertal testicular tissues, when autologously grafted under the back skin or scrotal skin of castrated pubertal rhesus macaques, matured to produce functional sperm ^[49]. Not only did the grafts grow and produce testosterone over an 8- to 12-month observation period, but complete spermatogenesis was also achieved in all grafts. Remarkably, the sperm derived from these grafts could fertilize rhesus oocytes, resulting in preimplantation embryo development, pregnancy, and even the birth of a healthy female baby. This breakthrough suggests that testicular tissue grafting holds significant promise for preserving the fertility of prepubertal patients undergoing gonadotoxic therapies.

So far, testicular tissues from more than twenty mammalian species have undergone xenografting. Impressively, in most of these species, both the spermatogenic and steroidogenic functions of the testicular tissue are re-established in the grafts. The technique has shown potential, achieving complete spermatogenesis in the most species used ^[50]^[51]. Going one step further, researchers have successfully regenerated functional testicular tissue by grafting testicular cells in suspension isolated from neonatal porcine or rodent testes onto mouse hosts ^[52]. These transplanted cells autonomously reorganized both the spermatogenic and interstitial compartments of the testis, producing functional haploid germ cells. This groundbreaking discovery offers a novel in vivo system to study mammalian spermatogenesis and testicular morphogenesis, providing an accessible platform for further investigations into these processes.

2.6. Cryopreservation Techniques for Xenotransplantations

Animals have played an instrumental role in advancing the field of testis tissue cryopreservation. Through systematic cryo-banking of their reproductive tissues, researchers have refined techniques that enhance reproductive management with the unique contribution of varying cryopreservation methods tailored to species-specific needs. As an example of species-specific variations in cryopreservation requirements during slow freezing, mandrill and marmoset testicular tissues are effectively preserved using a medium with 10% DMSO and 80% FBS. In comparison, chimpanzee tissues only require 20% DMSO without FBS ^[53]. On the contrary, fast freezing can be a better option for wild ungulates to cause less damage to sperm cells recovered from testicular tissues ^[53].

Several cryopreservation protocols have been designed tailored to specific species, notably humans, monkeys, and other primates. A common element across these protocols is the use of cryoprotectants, with dimethyl sulfoxide (DMSO) ^[54]^[55] and ethylene glycol (EG), reference ^[54] being the most prevalent, albeit in varying concentrations. The precision in cooling and warming rates is consistently emphasized, given their essential role in successful tissue preservation. Culture media, like MEM or DMEM, often supplemented with fetal bovine serum (FBS) or other additives, are utilized in some protocols. Using bovine fetal serum (BFS) and other xenogeneic additives in the cryopreservation of human testicular tissue raises several considerations. Bovine fetal serum is a rich supplement that provides essential growth factors, proteins, and hormones that can support the viability and functionality of cells during the cryopreservation process. However, its use in human tissue preservation might introduce concerns about potential cross-species contaminants, immunogenic reactions, and the ethical implications of sourcing BFS ^[56].

Patra et al. (2021) discuss the methodologies employed in the cryopreservation of human testicular tissues ^[55]. Slow freezing emerges as a favored method, with two distinct approaches: uncontrolled and controlled. The former is cost-effective, requiring minimal equipment and cryogenic agents, and is time-efficient. However, controlled slow freezing is more prevalent due to its consistent outcomes. This approach, though, confronts challenges like varied cryobiological properties of cell types and the extracellular matrix in tissue fragments and potential cytotoxic effects from prolonged exposure to cryoprotective agents.

Conversely, rapid freezing, which minimizes cell dehydration, has been less successful. It often results in extensive cryoinjuries due to unpredictable intracellular ice formation, leading to a high rate of cell death. As a result, its application in preserving testicular cells and tissues is limited.

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Pedro M. Aponte

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