In vitro spermatogenesis

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In vitro spermatogenesis is the process of creating male gametes (spermatozoa) outside of the body in a culture system. The process could be useful for fertility preservation, infertility treatment and may further develop the understanding of spermatogenesis at the cellular and molecular level.  

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Spermatogenesis is a highly complex process and artificially rebuilding it in vitro is challenging. [1] These include creating a similar microenvironment to that of the testis as well as supporting endocrine and paracrine signalling, and ensuring survival of the somatic and germ cells from spermatogonial stem cells (SSCs) to mature spermatozoa. [2]

Different methods of culturing can be used in the process such as isolated cell cultures, fragment cultures and 3D cultures [1]

Culture techniques

Isolated cell cultures

Cell cultures can include either monocultures, where one cell population is cultured, or co-culturing systems, where several cell lines (must be at least two) can be cultured together. [3] Cells are initially isolated for culture by enzymatically digesting the testis tissue to separate out the different cell types for culture [4] The process of isolating cells can lead to cell damage. [5]

The main advantage of monoculture is that the effect of different influences on one specific cell population of cells can be investigated.[ citation needed ] Co-culture allows for the interactions between cell populations to be observed and experimented on, which is seen as an advantage over the monoculture model. [3]

Isolated cell culture, specifically co-culture of testis tissue, has been a useful technique for examining the influences of specific factors such as hormones or different feeder cells on the progression of spermatogenesis in vitro.[ citation needed ] For example, factors such as temperature, feeder cell influence and the role of testosterone and follicle-stimulating hormone (FSH) have all been investigated using isolated cell culture techniques. [3]

Studies have concluded that different factors can influence the culture of germ cells e.g. media, growth factors, hormones and temperature. For example, when culturing immortalized mouse germ cells at temperatures of 35, 37 and 29°C, these cells proliferate most rapidly at the highest temperature and least rapidly at the lowest but there were varying levels of differentiation. At the highest temperature no differentiation were detected, some was seen at 37°C and some early spermatids appearing at 32°C. [3] Isolated cell culture technique has been successfully used for in vitro production of sperm using mouse as an animal model. [6]

Investigations of appropriate feeder cells concluded that a variety of cells could encourage development of germ cells such as Sertoli cells, Leydig cells and peritubular myoid cells but the most essential is Sertoli cells, but Leydig and peritubular myoid cells both contribute to the microenvironment that encourage stem cells to remain pluripotent and self renew in the testis. [7]

Testes fragment cultures

This image shows the difference in process between organ culture and cell culture. Testis Organ Culture Vs Cell Culture.jpg
This image shows the difference in process between organ culture and cell culture.

In fragment cultures, the testis is removed and fragments of tissue are cultured in supplemental media containing different growth factors to induce spermatogenesis and form functional gametes. [2] The development of this culture technique has taken place mainly with the use of animal models e.g. mice or rat testis tissue.

The advantage of using this method is that it maintains the natural spatial arrangement of the seminiferous tubules. However, hypoxia is a recurring problem in these cultures where the low oxygen supply hinders the development and maturation of spermatids (significantly more in adult than immature testis tissues). [2] Other challenges with this type of culture include maintaining the structure of the seminiferous tubules which makes it more difficult for longer-term cell cultures as the tissue structures can flatten out making it hard to work with. [7] To resolve some of these issues, 3D cultures can be used.

In 2012, mature spermatozoa capable of fertilization was isolated from in vitro culture of immature mouse testis tissue. [8]

3D cultures

3D cultures use sponge, models or scaffolds that resemble the elements of the extracellular matrix to achieve a more natural spatial structure of the seminiferous tubules and to better represent the tissues and the interaction between different cell types in an ex vivo experiment. Different components of the extracellular matrix such as collagen, agar and calcium alginate are commonly used to form the gel or scaffold which can provide oxygen and nutrients. [3] To propagate 3D cultures, testicular cell cultures are imbedded into the porous sponge/scaffold and allowed to colonise the structure which can then survive for several weeks to allow spermatogonia to differentiate and mature into spermatozoa.

In addition, shaking 3D cultures during the seeding process allows for an increased oxygen supply which helps overcome the issue of hypoxia and so improves the lifespan of cells. [3]

In contrast to monocultures, fragment/3D cultures are able to establish in vitro conditions that can somewhat resemble the testicular microenvironment to allow a more accurate study of the testicular physiology and its associations with the in vitro development of sperm cells. [3]

Future implications

Scientific

The ability to recapitulate spermatogenesis In vitro provides a unique opportunity to study this biological process through oftentimes cheaper and faster method of research than in vivo work. Observation is often easier in vitro, as the targeted cells are mostly isolated and immobile. Another significant advantage of in vitro research is the ease with which environmental factors can be changed and monitored. There are also techniques which are not practical or feasible in vivo which can now be explored. [8]

In vitro work is not without its own challenges. For example, one loses the natural structure provided by the in vivo tissue, and thus cell connections which could be important to the function of the tissue. [2]

Clinical

While rodent spermatogenesis is not identical to its human counterpart, especially due to the high evolution rate of the male reproductive tract, these techniques are a solid starting point for future human applications. [8]

Various categories of infertile men may benefit from advances in these techniques, especially those with a lack of viable gamete production. These men cannot benefit, for example, from sperm extraction techniques, and currently have little to no options for producing genetic descendants. [9]

Notably, males who have undergone chemo/radiotherapy prepubertally may benefit from in vitro spermatogenesis. [1] These people did not have the option to cryopreserve viable sperm before their procedure, and thus the ability to generate genetically descended sperm later in life is invaluable. Possible methods that could be applied (to this and other groups) are induction of spermatogenesis in testis samples taken prepubertally, or, if these samples are not available/viable, new methods that manipulate stem cell differentiation could produce SSCs 'from scratch', using adult stem cell samples. [8]

An alternative method is to graft preserved tissue back onto adult cancer survivors, however this comes with operational risks, as well as a risk of reintroducing malignant cells. [10] Even if using this method however, in vitro spermatogenesis advances would allow for sample expansion and observation to better ensure quality and quantity of graft tissue. [7]

In those with healthy or preserved SSCs but without a cellular environment to support them, in vitro spermatogenesis could be used following transplant of the SSCs into healthy donor tissue. [7]

Another group that could be helped by in vitro spermatogenesis are those with any form of genetic impediment to sperm production. Those with no viable SSC development are an obvious target, but also those with varying levels of spermatogenic arrest; previously their underdeveloped germ cells have been injected into oocytes, however this has a success rate of only 3% in humans. [7]

Finally, in vitro spermatogenesis using animal or human cells can be used to assess the effects and toxicity of drugs before in vivo testing. [3]

Related Research Articles

<span class="mw-page-title-main">Spermatozoon</span> Motile sperm cell

A spermatozoon is a motile sperm cell produced by male animals relying on internal fertilization. A spermatozoon is a moving form of the haploid cell that is the male gamete that joins with an ovum to form a zygote.

<span class="mw-page-title-main">Testicle</span> Internal organ in the male reproductive system

A testicle or testis is the gonad in all male bilaterians, including humans, and is homologous to the ovary in females. Its primary functions are the production of sperm and the secretion of androgens, primarily testosterone.

<span class="mw-page-title-main">Gametogenesis</span> Biological process

Gametogenesis is a biological process by which diploid or haploid precursor cells undergo cell division and differentiation to form mature haploid gametes. Depending on the biological life cycle of the organism, gametogenesis occurs by meiotic division of diploid gametocytes into various gametes, or by mitosis. For example, plants produce gametes through mitosis in gametophytes. The gametophytes grow from haploid spores after sporic meiosis. The existence of a multicellular, haploid phase in the life cycle between meiosis and gametogenesis is also referred to as alternation of generations.

<span class="mw-page-title-main">Germ cell</span> Gamete-producing cell

A germ cell is any cell that gives rise to the gametes of an organism that reproduces sexually. In many animals, the germ cells originate in the primitive streak and migrate via the gut of an embryo to the developing gonads. There, they undergo meiosis, followed by cellular differentiation into mature gametes, either eggs or sperm. Unlike animals, plants do not have germ cells designated in early development. Instead, germ cells can arise from somatic cells in the adult, such as the floral meristem of flowering plants.

<span class="mw-page-title-main">Spermatogenesis</span> Production of sperm

Spermatogenesis is the process by which haploid spermatozoa develop from germ cells in the seminiferous tubules of the testicle. This process starts with the mitotic division of the stem cells located close to the basement membrane of the tubules. These cells are called spermatogonial stem cells. The mitotic division of these produces two types of cells. Type A cells replenish the stem cells, and type B cells differentiate into primary spermatocytes. The primary spermatocyte divides meiotically into two secondary spermatocytes; each secondary spermatocyte divides into two equal haploid spermatids by Meiosis II. The spermatids are transformed into spermatozoa (sperm) by the process of spermiogenesis. These develop into mature spermatozoa, also known as sperm cells. Thus, the primary spermatocyte gives rise to two cells, the secondary spermatocytes, and the two secondary spermatocytes by their subdivision produce four spermatozoa and four haploid cells.

<span class="mw-page-title-main">Seminiferous tubule</span> Location of meiosis and creation of spermatozoa

Seminiferous tubules are located within the testicles, and are the specific location of meiosis, and the subsequent creation of male gametes, namely spermatozoa.

<span class="mw-page-title-main">Sertoli cell</span> Cells found in human testes which help produce sperm

Sertoli cells are a type of sustentacular "nurse" cell found in human testes which contribute to the process of spermatogenesis as a structural component of the seminiferous tubules. They are activated by follicle-stimulating hormone (FSH) secreted by the adenohypophysis and express FSH receptor on their membranes.

Capacitation is the penultimate step in the maturation of mammalian spermatozoa and is required to render them competent to fertilize an oocyte. This step is a biochemical event; the sperm move normally and look mature prior to capacitation. In vivo, capacitation occurs after ejaculation, when the spermatozoa leave the vagina and enter the upper female reproductive tract. The uterus aids in the steps of capacitation by secreting sterol-binding albumin, lipoproteins, and proteolytic and glycosidasic enzymes such as heparin.

<span class="mw-page-title-main">Spermatocyte</span> Sperm precursor cell that undergoes meiosis

Spermatocytes are a type of male gametocyte in animals. They derive from immature germ cells called spermatogonia. They are found in the testis, in a structure known as the seminiferous tubules. There are two types of spermatocytes, primary and secondary spermatocytes. Primary and secondary spermatocytes are formed through the process of spermatocytogenesis.

<span class="mw-page-title-main">Spermatogonium</span> Undifferentiated male germ cell

A spermatogonium is an undifferentiated male germ cell. Spermatogonia undergo spermatogenesis to form mature spermatozoa in the seminiferous tubules of the testicles.

<span class="mw-page-title-main">Blood–testis barrier</span> Physical barrier between the blood vessels and the seminiferous tubules of animal testes

The blood–testis barrier is a physical barrier between the blood vessels and the seminiferous tubules of the animal testes. The name "blood-testis barrier" is misleading as it is not a blood-organ barrier in a strict sense, but is formed between Sertoli cells of the seminiferous tubule and isolates the further developed stages of germ cells from the blood. A more correct term is the Sertoli cell barrier (SCB).

Stem-cell niche refers to a microenvironment, within the specific anatomic location where stem cells are found, which interacts with stem cells to regulate cell fate. The word 'niche' can be in reference to the in vivo or in vitro stem-cell microenvironment. During embryonic development, various niche factors act on embryonic stem cells to alter gene expression, and induce their proliferation or differentiation for the development of the fetus. Within the human body, stem-cell niches maintain adult stem cells in a quiescent state, but after tissue injury, the surrounding micro-environment actively signals to stem cells to promote either self-renewal or differentiation to form new tissues. Several factors are important to regulate stem-cell characteristics within the niche: cell–cell interactions between stem cells, as well as interactions between stem cells and neighbouring differentiated cells, interactions between stem cells and adhesion molecules, extracellular matrix components, the oxygen tension, growth factors, cytokines, and the physicochemical nature of the environment including the pH, ionic strength and metabolites, like ATP, are also important. The stem cells and niche may induce each other during development and reciprocally signal to maintain each other during adulthood.

Female sperm can refer to either:

  1. A sperm which contains an X chromosome, produced in the usual way in the testicles, referring to the occurrence of such a sperm fertilizing an egg and giving birth to a female.
  2. A sperm which artificially contains genetic material from a female.

Testicular Immunology is the study of the immune system within the testis. It includes an investigation of the effects of infection, inflammation and immune factors on testicular function. Two unique characteristics of testicular immunology are evident: (1) the testis is described as an immunologically privileged site, where suppression of immune responses occurs; and, (2) some factors which normally lead to inflammation are present at high levels in the testis, where they regulate the development of sperm instead of promoting inflammation.

Gonocytes are the precursors of spermatogonia that differentiate in the testis from primordial germ cells around week 7 of embryonic development and exist up until the postnatal period, when they become spermatogonia. Despite some uses of the term to refer to the precursors of oogonia, it was generally restricted to male germ cells. Germ cells operate as vehicles of inheritance by transferring genetic and epigenetic information from one generation to the next. Male fertility is centered around continual spermatogonia which is dependent upon a high stem cell population. Thus, the function and quality of a differentiated sperm cell is dependent upon the capacity of its originating spermatogonial stem cell (SSC).

FNA mapping is an application of fine-needle aspiration (FNA) to the testis for the diagnosis of male infertility. FNA cytology has been used to examine pathological human tissue from various organs for over 100 years. As an alternative to open testicular biopsy for the last 40 years, FNA mapping has helped to characterize states of human male infertility due to defective spermatogenesis. Although recognized as a reliable, and informative technique, testis FNA has not been widely used in U.S. to evaluate male infertility. Recently, however, testicular FNA has gained popularity as both a diagnostic and therapeutic tool for the management of clinical male infertility for several reasons:

  1. The testis is an ideal organ for evaluation by FNA because of its uniform cellularity and easy accessibility.
  2. The trend toward minimally invasive procedures and cost-containment views FNA favorably compared to surgical testis biopsy.
  3. The realization that the specific histologic abnormality observed on testis biopsy has no definite correlation to either the etiology of infertility or to the ability to find sperm for assisted reproduction.
  4. Assisted reproduction has undergone dramatic advances such that testis sperm are routinely used for biological pregnancies, thus fueling the development of novel FNA techniques to both locate and procure sperm.

Takehiko Ogawa is a Japanese urologist and developmental biologist, known for his pioneer research on in vitro spermatogenesis. He is Professor of Proteomics at Graduate School of Medical Life Science, Yokohama City University.

<span class="mw-page-title-main">Spermatogonial stem cell</span> Spermatogonium that does not differentiate into a spermatocyte

A spermatogonial stem cell (SSC), also known as a type A spermatogonium, is a spermatogonium that does not differentiate into a spermatocyte, a precursor of sperm cells. Instead, they continue dividing into other spermatogonia or remain dormant to maintain a reserve of spermatogonia. Type B spermatogonia, on the other hand, differentiate into spermatocytes, which in turn undergo meiosis to eventually form mature sperm cells.

<span class="mw-page-title-main">Peritubular myoid cell</span> Smooth muscle cell found in testis

A peritubular myoid (PTM) cell is one of the smooth muscle cells which surround the seminiferous tubules in the testis. These cells are present in all mammals but their organization and abundance varies between species. The exact role of PTM cells is still somewhat uncertain and further work into this is needed. However, a number of functions of these cells have been established. They are contractile cells which contain actin filaments and are primarily involved in transport of spermatozoa through the tubules. They provide structural integrity to the tubules through their involvement in laying down the basement membrane. This has also been shown to affect Sertoli cell function and PTM cells also communicate with Sertoli cells through the secretion of growth factors and ECM components. Studies have shown PTM cells to be critical in achieving normal spermatogenesis. Overall, PTM cells have a role in both maintaining the structure of the tubules and regulating spermatogenesis through cellular interaction.

Cryopreservation of testicular tissue is an experimental method being used to preserve fertility in pre-pubescent males, or males who cannot produce sperm, to allow them the option of having biological children.

References

  1. 1 2 3 Ibtisham, Fahar; Honaramooz, Ali (18 March 2020). "Spermatogonial Stem Cells for In Vitro Spermatogenesis and In Vivo Restoration of Fertility". Cells. 9 (3): 745. doi: 10.3390/cells9030745 . PMC   7140722 . PMID   32197440.
  2. 1 2 3 4 Reuter, Karin; Schlatt, Stefan; Ehmcke, Jens; Wistuba, Joachim (2012-10-01). "Fact or fiction: In vitro spermatogenesis". Spermatogenesis. 2 (4): 245–252. doi:10.4161/spmg.21983. ISSN   2156-5554. PMC   3521746 . PMID   23248765.
  3. 1 2 3 4 5 6 7 8 Galdon, Guillermo; Atala, Anthony; Sadri-Ardekani, Hooman (2016-04-23). "In Vitro Spermatogenesis: How Far from Clinical Application?". Current Urology Reports. 17 (7): 49. doi:10.1007/s11934-016-0605-3. ISSN   1527-2737. PMID   27107595. S2CID   35666011.
  4. ↑ Ibtisham, Fahar; Zhao, Yi; Wu, Jiang; Nawab, Aamir; Mei, Xiao; Li, GuangHui; An, Lilong (March 2019). "The optimized condition for the isolation and in vitro propagation of mouse spermatogonial stem cells" (PDF). Biologia Futura. 70 (1): 79–87. Bibcode:2019BioFu..70...79I. doi:10.1556/019.70.2019.10. PMID   34554427. S2CID   146104690.
  5. ↑ Hunter, Damien; Anand-Ivell, Ravinder; Danner, Sandra; Ivell, Richard (2012-01-01). "Models of in vitro spermatogenesis". Spermatogenesis. 2 (1): 32–43. doi:10.4161/spmg.19383. ISSN   2156-5554. PMC   3341244 . PMID   22553488.
  6. ↑ Ibtisham, Fahar; Wu, Jiang; Xiao, Mei; An, Lilong; Banker, Zachary; Nawab, Aamir; Honaramooz, Ali (2021-01-01). "In vitro production of haploid germ cells from murine spermatogonial stem cells using a two-dimensional cell culture system". Theriogenology. 162 (2021): 84–94. doi:10.1016/j.theriogenology.2020.12.024. PMID   33450717. S2CID   231623654.
  7. 1 2 3 4 5 6 Ibtisham, Fahar; Wu, Jiang; Xiao, Mei; An, Lilong; Banker, Zachary; Nawab, Aamir; Zhao, Yi; Li, Guanghui (2017-09-12). "Progress and future prospect of in vitro spermatogenesis". Oncotarget. 8 (39): 66709–66727. doi:10.18632/oncotarget.19640. ISSN   1949-2553. PMC   5630449 . PMID   29029549.
  8. 1 2 3 4 Song, Hye-Won; Wilkinson, Miles F. (2012-10-01). "In vitro spermatogenesis". Spermatogenesis. 2 (4): 238–244. doi:10.4161/spmg.22069. ISSN   2156-5554. PMC   3521745 . PMID   23248764.
  9. ↑ Fattahi, Amir; Latifi, Zeinab; Ghasemnejad, Tohid; Nejabati, Hamid Reza; Nouri, Mohammad (July 2017). "Insights into in vitro spermatogenesis in mammals: Past, present, future". Molecular Reproduction and Development. 84 (7): 560–575. doi: 10.1002/mrd.22819 . ISSN   1098-2795. PMID   28436137.
  10. ↑ Ibtisham, Fahar; Awang-Junaidi, Awang Hazmi; Honaramooz, Ali (May 2020). "The study and manipulation of spermatogonial stem cells using animal models". Cell and Tissue Research. 380 (2): 393–414. doi:10.1007/s00441-020-03212-x. PMID   32337615. S2CID   216146411.
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