Spermatogenesis in immature mammals

Abstract

Mammalian spermatogenesis has been studied extensively as a prime theme of male reproductive biology, especially for germ cell production, fertilization and development. Investigation of spermatogenesis has provided us with the opportunity to both study the male germ line stem cells and generate the transgenic animals. Spermatogenesis is conducted in the seminiferous tubules, which end in the rete testis. The organization of spermatogenesis means that the spermatogonia are uniformly distributed around the seminiferous tubules. The pubertal establishment and mature maintenance of spermatogenesis requires precursor cells. In bull testes at 4 weeks postnatal, gonocyte migration occurs and differentiated spermatogonia are recognized after 8 weeks. Within the period of 4–8 weeks of age, spermatogonial stem cell conversion and niche formation must occur. Spermatogonial stem cells are the only cells that can undergo self‐renewal in spermatogenesis. Spermatogonial stem cell transplantation can potentially contribute to studies of gene expression during spermatogenesis and provide genetic progress in domestic animals. Bull spermatogonial stem cells have been demonstrated to be capable of colonizing recipient mouse seminiferous tubules. (Reprod Med Biol 2007; 6: 139–149)

INTRODUCTION

SPERMATOGENESIS IN MAMMALIAN testis is one of the most productive processes in the male. In immature mammals, both proliferation and differentiation of germ cells will result in the first wave of spermatogenesis and for the future continuous sperm production of mature mammalian testis. Sustainable sperm production in the male relies on spermatogonial stem cells having the ability to undergo both actions of production of daughter progeny committed to the spermatogenic pathway and self‐renewal proliferation. In mice, spermatogonial stem cells have been genetically modified in vitro and have resulted in transgenic offspring being sired by transplanted recipients. ¹ The important aspects of spermatogonial stem cells has focused interest on their use in reproductive technologies such as spermatogonial stem cell transplantation and transgenesis. The ability to culture spermatogonial stem cells may give us a new approach through male gametes to introduce transgenes into domestic animals. Spermatogenesis is a highly organized and complex process occurring in the testis that involves the interaction of both germ and somatic cells. The germ cell contributes to the proliferation and differentiation of many cell types, beginning with diploid spermatogonial stem cells and ending with haploid spermatozoa. In contrast, the somatic cell contributes to steroid biosynthesis by Leydig cells and the nurse function of Sertoli cells.

Development of immature testis includes a time period in which both somatic and germ cells undergo proliferation and differentiation that will result in the first wave of spermatogenesis and set the framework for sustainable sperm production in mature testis. The ultimate application of spermatogonial stem cell cultures and transplantation technology in farm animals may be the ability to generate transgenic offspring. To accomplish this, one of several challenges is the ability to culture spermatogonial stem cells from farm animals. Once a signal stimulates the initial division and differentiation of the diploid spermatogonial stem cells into spermatogonia, spermatocytogenesis occurs leading to the expansion of a cohort of genetically identical germ cells capable of becoming spermatocytes. Although critically important for the production of sperm, spermatogonial stem cells have been difficult to study because of the small number of these cells in the testis and the challenges associated with identifying, culturing and assaying their biological activity. Manipulation of germ cells provides an opportunity to study the mechanisms underlining continuation of the germline and to develop novel technologies for germline modification. Recent research with mouse spermatogonial stem cells in the mouse ² , ³ , ⁴ has been demonstrated to be the important problem in a new step of developmental and reproductive application for biology, animal production and medicine.

In this review, we present, in brief, morphological analysis of spermatogenesis and gene expression, and a functional analysis of germ cells in immature mice and bulls.

MORPHOLOGICAL ANALYSIS OF SPERMATOGENESIS IN IMMATURE MAMMALS

SPERMATOGENESIS IS CONDUCTED in seminiferous tubules, which end in the rete testis. This process is uniform within seminiferous tubules and spermatids are lined evenly along the lumen and released all around the lumen. The organization of spermatogenesis also means that the spermatogonia are uniformly distributed around the seminiferous tubules. The pubertal establishment and mature maintenance of spermatogenesis requires that the precursor cells not be positioned within the tubule asymmetrically. Transplantation of a single stem cell spermatogonium ² will result initially in asymmetrical spermatogenesis. ⁵

Localization of germ cells and Sertoli cells in immature (from day 0 to day 18) mouse seminiferous tubules was examined (Fig. 1). Mouse gonocytes migrate to the basement membrane and undergo a conversion to stem cells from days 0 to 5 postnatally, where spermatogonial stem cell niches are formed, from which all differentiating germ cells will arise. ⁶ Spermatogonial stem cells are capable of movement. Spermatogenesis is established at the rate of approximately 60 µm/day along the length of the seminiferous tubule after transplantation ⁷ into an infertile recipient. The capability for stem cells to move and the uniform organization of spermatogenesis around seminiferous tubules suggest that some regulatory influence must govern the even distribution of spermatogenic cells during normal spermatogenesis. It has been suggested that the distribution of primitive classes of spermatogonia throughout most of the spermatogenic cycle is not random. ⁸ Type A spermatogonia that are believed to be stem cells are called A single (A_s), proliferative cells are called A paired (A_pr) and A aligned (A_al), and differentiated cells are called A₁, A₂, A₃, A₄. The spermatogonia are mobile and have demonstrated the ability to cyclically position themselves at periodic intervals along the seminiferous tubule to ensure their progeny are distributed evenly and uniformly around the seminiferous tubule. Several results allowed us to show that specific type A spermatogonia were not randomly positioned within the seminiferous tubules at certain stages of the cycle of the seminiferous epithelium. An interesting finding is that a non‐random distribution of spermatogonia could only be maintained if seminiferous tubules remained in a relatively constant relationship to one another. A study in the mouse strongly suggests that their seminiferous tubules do not move relative to one another or rotate in vivo. The sites around the tubule provide two to eight relatively evenly spaced foci from which their progeny can spread laterally.

Figure 1.

Localization of germ cells and Sertoli cells (S) in immature (a, day 0; b, day 6; c, day 8; d, day 18) mouse seminiferous tubules. G, gonocytes; DA, dark A spermatogonia; LA, light A spermatogonia; A, A spermatogonia; B, B spermatogonia; L, leptotene spermatocyte; P, pachytene spermatocyte.

Sertoli cells of a particular species support a characteristic number of germ cells. ⁹ This number has never been shown to increase by experimental manipulation beyond that seen in normal animals. Thus, it appears that the germ cells, up to preleptotene spermatocytes, are rather evenly distributed around the seminiferous tubule. They move centripetally between Sertoli cells ¹⁰ and then, as elongating spermatids, form an exclusive association with a single Sertoli cell. ¹¹ The periodic location of spermatogonia near the interstitium serves to position spermatogonia at evenly spaced foci, from which further cell division occurs that will later spread the progeny of type A spermatogonia evenly around the base of the seminiferous tubule. It appears that the distribution of the A_s to A_al spermatogonia is located primarily at the interstitium, but the number of cells is regulated in a different manner. In the mitotic divisions from A₂ to A₄ spermatogonia, a mechanism limits the number of spermatogonia in particular regions of the tubule, based on their density. This regulation in mice appears to occur via a prosurvival protein (Bclx‐L ¹² ) and a pro‐apoptotic protein (Bax ¹³ ). Thus, mechanisms are in place to regulate both the numbers and spatial distribution of early germ cells in mice.

Testosterone is a possible candidate as an attractant to spermatogonia via stimulation of nearby somatic cells, given that no testosterone receptors are found in spermatogonia. At first examination, this possibility seems unlikely because considerable information suggests that increased testosterone levels increase spermatogonial apoptotic activity and that lowered levels do the opposite. ¹⁴ , ¹⁵ However, on further examination, the suppression of spermatogonial apoptosis should not be confused with signaling for cells to migrate. Leydig cells are known to produce other factors that may be attractants for spermatogonia. ¹⁶ Chiarini‐Garcia and Russell demonstrated that spermatogonia migrate within the seminiferous epithelium. ⁸ Other recent studies have also demonstrated this migratory capability of spermatogonia. For example, a study using radiation depletion of the seminiferous epithelium has shown that when most spermatogonial stem cells are depleted, repopulation of the tubule occurs as the few remaining spermatogonia undergo mitosis and extend along the length of the seminiferous tubule. ¹⁷ The movements referred to above are accomplished by spermatogonial stem cells. It has been suggested that the successors of A₁ to A₃ cells, preleptotene–leptotene spermatocytes move centripetally at the time they enter the intermediate compartment of the testis and that Sertoli cells are helpful in relocating preleptotene–leptotene spermatocytes; ¹⁰ however, the data do not indicate if the surrounding Sertoli cells are active in helping spermatogonia move. The A_s, A_pr and A_al populations are not an equal numerical distribution of cell types. In most stages, the A_al population dominates in terms of cell numbers. ⁸ The more primitive of the spermatogonia, the A_s to A_al cells, are preferentially positioned at specific sites along the periphery of the seminiferous tubule. The apparent preferential location of these cells reflects the need for an evenly spaced distribution of selective precursor type A spermatogonia.

In immature mice spermatogenesis, some results suggested that specific type A spermatogonia were not randomly positioned within the seminiferous tubules at certain stages of the cycle of the seminiferous epithelium and spermatogenic wave. An interesting finding is that a non‐random distribution of spermatogonia could only be maintained if seminiferous tubules remained in a relatively constant relationship to one another. As mentioned above, results from immature mouse spermtogenesis strongly suggest that their seminiferous tubules do not move relative to one another or rotate in vivo. In mouse testis from days 0 to 5 postnatally, gonocytes migrate to the basement membrane and undergo a conversion to stem cells, where spermatogonial stem cell niches are formed, from which all differentiating germ cells will begin. ⁶ In the bull testis, the migration, conversion and niche formation occurs over a period of months. Correlations between the testis weight and body weight of immature bulls were examined (Fig. 2). Body weight increased with age. At 4, 8, 12 and 28 weeks of age, body weight was 55, 80, 100 and 180 kg, respectively. At 4 weeks postnatally, gonocyte migration occurs, and differentiated spermatogonia are not present until 8 weeks of age. ¹⁸ Therefore, within this period of 4–8 weeks of age, spermatogonial stem cell conversion and niche formation must occur. Oatley et al. have demonstrated that transplantation of germ cells from bull calves older than 4 weeks of age results in the best colonization in recipient mouse seminiferous tubules, ¹⁹ further indicating that spermatogonial stem cell conversion is not complete until after 4 weeks of age in the bull.

Figure 2.

Correlation between testis weight and body weight of immature bulls. At 4, 8, 12 and 28 weeks of age, body weight was 55, 80, 100 and 180 kg, respectively.

Currently, spermatogonial stem cells can only be unequivocally identified on a functional basis by their ability to colonize recipient seminiferous tubules following transplantation. Development of in vitro culture techniques that support the survival, proliferation and differentiation of spermatogonial stem cells will aid in the ability to study them as well as modify their genome. Culture of mouse spermatogonial stem cells in vitro has been demonstrated using feeder layers for periods extending up to 4 months. ²⁰ In vitro maturation of bovine gonocytes through meiosis has been reported using calcium–alginate encapsulation; ²¹ however, stem cell activity using this method has not been investigated. The ability to modify bovine spermatogonial stem cells would be facilitated by use of an in vitro culture system that supports their survival and proliferation. Because spermatogonial stem cell transplantation between bulls is not possible, a bioassay model to evaluate bovine cells has been developed. Bull germ cells are capable of colonization and proliferation in recipient immunodeficient mouse seminiferous tubules. ¹⁹ , ²² This bioassay can be used to evaluate culture and transfection techniques with bovine spermatogonial stem cells. Oatley et al. demonstrated that a culture system was devised where Sertoli, Leydig and spermatogonial stem cell interaction would be maintained. Their results demonstrated that Sertoli cells remain viable for at least 2 weeks during tissue explant culture based on their ability to express stem cell factor. This ligand secreted by Sertoli cells has been demonstrated to be essential in regulating spermatogonial proliferation in the testis through c‐kit present on differentiated spermatogonia. ²³ Both stem cell factor and testosterone secretion in the testis are known to be under the control of follicle stimulating hormone (FSH) and luteinizing hormone (LH), respectively. Development of in vitro culture systems for bovine spermatogonial stem cells has been a challenge because of the lack of a functional system to evaluate their presence in a cell suspension. Cross‐species transplantation of bovine cells into mouse testes is the only functional method for evaluating the survival and proliferation of bovine spermatogonial cells in culture. ¹⁹ , ²² The demonstration that explant cultured bovine cells could form round cell colonies in recipient mouse seminiferous tubules indicates the survival of spermatogonial stem cells. The observed round cell morphology of the colonized bovine cells is consistent with spermatogonial stem cell colonization as previously observed in other cross‐species transplantations into recipient mouse testes. ²² , ²⁴ , ²⁵ It is unlikely that the colonizing donor cells in recipient mouse seminiferous tubules were differentiated germ cells or somatic cells.

The exact mechanism of the proliferation remains unknown, but speculation could be made that Sertoli and interstitial cells are inducing proliferation in response to a lack of meiotic germ cells in the seminiferous epithelium. An explanation of spermatogonial stem cell proliferation is related to the age of the bull. At 1–2 months of age, bull gonocytes have just migrated to the basement membrane and are likely to be undergoing conversion to stem cells and forming niches. Culture of gonocytes from neonatal bull calves in a calcium–alginate encapsulation system has been demonstrated to support the maturation of round spermatids after 10 weeks of culture. ²¹ In an in vivo situation, haploid germ cells of a normal bull are not present until 24–28 weeks of age. ¹⁸ In vitro maturation in the bovine supports the theory that the removal of germ cells and somatic cells from a prepubertal animal stimulates spontaneous germ cell actions. Many reports have demonstrated an inhibition of spermatogenesis at higher temperatures in bulls and rodents as a result of cryptorchidism. ²⁶ Examination of the cultured tissues with lower temperatures revealed maintenance of seminiferous tubular structure and a significant increase in germ cell numbers compared with fresh tissue. Recently, postnatal spermatogenesis has been demonstrated in testicular tissue from several species xenografted onto the backs of recipient mice. ²⁷ , ²⁸ Although the first round of spermatogenesis in testicular tissue ectopically xenografted onto mice has not been reported in the bovine, ²⁹ spermatogenesis in testicular tissue is accelerated in mice, pigs, goats and primates. Successful establishment of spermatogenesis has required neonatal donors as a source of testicular tissue in previous studies. ²⁷ , ²⁸ , ²⁹ , ³⁰

During testis development of immature mammals, both the somatic and germ cells undergo stages of developmental change. In postnatal mice (days 1–5), the gonocytes undergo translocation to the basement membrane followed by conversion to spermatogonial stem cells. ³¹ , ³² , ³³ In bulls, this period is much longer, from day 0 to 16 weeks when differentiating spermatogonia are first recognized. ¹⁸ Sertoli cells appeared to maintain their functional abilities to produce growth factors. Maturation of male germ cells in the testis is dependent on interaction with Sertoli cells. Both testosterone and FSH are necessary for qualitative and quantitative spermatogenesis. ³⁴ , ³⁵ The exact mechanisms that control spermatogonial stem cell activity in the testis are unknown. Recently, a number of studies demonstrated the survival and proliferation of bovine spermatogonial stem cells during explant testis tissue culture over a 2‐week period.

During this early stage of postnatal testicular development, the somatic cell population consists of immature proliferative Sertoli and Leydig cells supporting spermatogenesis. In the postnatal mouse testis, Sertoli cell proliferation occurs from day 0 to day 10, similar to the time of differentiation. ³⁶ Oatley et al. hypothesized that testicular tissue from 12‐week‐old bulls would result in the most effective establishment of spermatogenesis following grafting because this is the age just before germ cell maturation into spermatogonia is initiated. During the neonatal phase of testicular development, somatic Sertoli and Leydig cells undergo proliferation and differentiation. Changes in both the somatic and germ cells during the neonatal period could have an impact on the establishment of spermatogenesis. Oatley et al. proposed that testicular tissue from 12‐week‐old bulls would be the ideal donor tissue and that 8‐week‐old donors provided the best testis tissue source for grafting. Lack of Sertoli cell maturation and attainment of normal differentiated function would result in an inability to support germ cell maturation. Previous studies have demonstrated an acceleration of the first round of postnatal spermatogenesis in neonatal testicular tissue of pigs, goats, mice and primates ectopically xenografted onto castrated recipient mice. ²⁷ , ²⁸ , ²⁹ A lack of observed elongated spermatids in 2‐week‐old neonatal bovine testicular tissue 24 weeks after grafting suggests that a comparable acceleration does not occur with the bovine. In the intact bull, elongated spermatids are first observed at 28 weeks postnatal age. ¹⁸ Testicular tissue at the ages of 12 and 16 weeks possibly contains a high percentage of seminiferous tubules with somatic and germ cell populations. The number of seminiferous tubules counted to determine the percentage of tubules containing differentiating germ cells varied slightly between groups because of the bull donor age and the differentiation of germ cells in grafts at removal.

Use of neonatal donors is of benefit because undifferentiated spermatogonia are the only germ cell type present and the population is enriched for spermatogonial stem cells. Understanding the establishment of spermatogenesis in grafted neonatal testicular tissue can lead to enhancement of sperm production. Oatley et al. demonstrated differences in the establishment of spermatogenesis in testicular tissue from bull calves at different neonatal stages of testicular differentiation ectopically xenografted onto recipient mice.

GERM CELL FUNCTIONS AND GENE EXPRESSION DURING SPERMATOGENESIS IN IMMATURE MICE

SPERMATOGENESIS IS MAINTAINED through continuous activity of spermatogonial stem cells. The spermatogonial stem cells reside in stem cell niches located on the basement membrane of seminiferous tubules and among the basal portions of Sertoli cells. ³⁷ , ³⁸ The crucial role of testicular stem cells is to maintain their own pool by self‐renewal and to provide germ cell progenitors that undergo mitotic cell divisions and, ultimately, differentiate into spermatozoa. ³⁷ , ³⁹ , ⁴⁰ , ⁴¹ , ⁴² Although the function of spermatogonial stem cells is crucial for the maintenance of spermatogenesis, relatively little is known about their biology, ³⁷ , ⁴² , ⁴³ mainly because studies of testicular stem cells in rodents are inhibited by the difficult discrimination between stem cells and differentiating spermatogonia. Only subtle morphological differences exist between spermatogonial stem cells (A_s), undifferentiated spermatogonia (A_pr and A_al) and differentiating A₁ through A₄ spermatogonia, ⁴⁴ and no specific markers have been described to distinguish unequivocally spermatogonial stem cells from the differentiating progeny. ³⁷ , ⁴¹ , ⁴⁵ , ⁴⁶ Furthermore, the low number of stem cells in the testes is an obstacle to the study of testicular stem cells.

CD9 and α6‐integrin and β1‐integrin are commonly found on stem cells, and these surface protein markers may facilitate interactions between stem cells and their niches located on tissue basement membranes. ¹⁴ , ⁴⁷ , ⁴⁸ Recently, glial cell line derived neurotrophic factor (GDNF) was proposed to be an important growth factor for communication between spermatogonia and Sertoli cells. The GDNF promotes the proliferation of undifferentiated spermatogonia both in vitro and in vivo. ³⁸ , ⁴² , ⁴⁵ , ⁴⁹ In the testis, GDNF is secreted by Sertoli cells ³⁸ , ⁴⁵ , ⁵⁰ and GDNF family receptor alfa 1 (GFRA1) is expressed by spermatogonial stem cells ⁴⁹ , ⁵⁰ , ⁵¹ , ⁵² and the GFRA1 receptor is expressed in undifferentiated spermatogonia. ⁴⁵ , ⁴⁹ Spermatogonial stem cells are the only cells that can undergo self‐renewal in spermatogenesis. Despite their importance in male reproduction, little is known about these stem cells. Mouse testes are estimated to contain only two or three stem cells per 10⁴ testis cells. ⁵³ The development of methods to purify these stem cells is essential. Purification of spermatogonial stem cells has been most successful in mice. ⁴³ Mouse spermatogonial stem cells can now be routinely isolated as follows. Shinohara et al. successfully enriched the stem cell population to 1 in 30 testis cells, ⁴³ and other researchers have achieved 700‐fold enrichment using a specific marker for undifferentiated spermatogonia. ⁵⁴ Magnetic activated cell sorting (MACS) has been used effectively for cell isolation in many tissues and organs. ⁵⁵ , ⁵⁶ The MACS has been shown to be a successful tool for the enrichment of spermatogonial stem cells, ⁵⁰ , ⁵⁷ and studies have shown that spermatogonial stem cells share some surface protein antigens that are also present on other stem cells. ⁴⁶ , ⁵⁸ The MACS is specifically useful for stem cell isolation because stem cell numbers are very low. The efficiency of MACS depends on the availability of specific surface markers on the membranes of the stem cells. Spermatogonial stem cells are the male specific germ cells and are found only in the postnatal testis. ⁴⁰ Buageaw et al. ⁵⁹ have confirmed GFRA1 expression in spermatogonial stem cells and have shown that GFRA1 antibodies can be used to obtain enriched fractions of spermatogonial stem cells using MACS. Their results indicate the existence of a distinct subpopulation of spermatogonial stem cells in the mouse pup testis that expresses a higher level of GFRA1. Shinohara et al. ⁴⁶ investigated for antigens that are expressed on spermatogonial stem cells and hypothesized that stem cells of many self‐renewing tissues share a common molecule. Shinohara et al. ⁴⁶ examined the expression of CD9, which is expressed on other types of stem cells. ⁴⁷ , ⁵⁹ Mouse testis cells were selected by the MACS and examined for the stem cell activity by spermatogonial transplantation. On the other hand, mouse spermatogonial stem cells also has been shown expressing CD9, Oct –4 and Stra8. ⁴⁵ , ⁵⁴ CD9 is a commonly expressed molecule on several types of stem cells and is a membrane protein with four transmembrane domains and is involved in some cell functions. ⁶⁰ , ⁶¹ , ⁶² In the testis, expression of CD9 is not specific to stem cells. Non‐specific expression of CD9 on stem cells was recognized from the results of immunohistochemical staining. CD9‐positive cells are identified using other cell‐specific markers. A new surface antigen on stem cells may improve the enrichment protocols in mice. CD9 may play a role in signal transduction and in cellular adhesion. ⁶³ Further functional study is required to investigate the role of CD9 in mammalian spermatogenesis. The expression of CD9 and c‐kit mRNA were examined using reverse transcriptase‐polymerase chain reaction (RT‐PCR) for days 2, 5, 8, 14, 24, 34 and for adult mice (Fig. 3). Stem cell purification in many self‐renewing tissues is based on cell selection using a fluorescence activated cell sorter (FACS) or centrifugation. ⁴³ Vitamin A deficient animals have only undifferentiated spermatogonia ⁶⁴ , ⁶⁵ and the cryptorchid operation enriches to 1 in 200 mouse spermatogonial stem cells. ⁶⁶ In addition, the number of stem cells in the neonatal testis is smaller than that in the adult testis. ³²

Figure 3.

Expression of CD9 and c‐kit mRNA by mouse testis. The expression of CD9 and c‐kit was examined using reverse transcriptase‐polymerase chain reaction for days 2, 5, 8, 14, 24, 34 and adult mice. β‐actin RNA was included in the total RNA as an external control. The values are means of the ratio of CD9 or c‐kit mRNA to β‐actin mRNA.

It was rescently suggested that the cell enrichment technique may be extended to other species; however, few surface markers limits application of the selection strategy to testis cells. It is possible to select neonatal gonocytes with high stem cell activity using micromanipulation. ⁶⁷ The other selection procedure used with stem cells is based on size and morphology. Spermatogonial stem cell transplantation has been reported showing that these cells can still attach to the basement membrane of mouse seminiferous tubules and proliferate in hamsters, cattle, pigs, primates and humans. ¹⁹ , ²² , ²⁴ , ²⁵ , ⁶⁸ , ⁶⁹ , ⁷⁰ , ⁷¹ Further investigations should be undertaken to identify new molecules on the stem cells.

GERM CELL FUNCTIONS AND GENE EXPRESSION DURING SPERMATOGENESIS IN IMMATURE BULLS

BASED ON PREVIOUS studies in mice, gene expression during spermatogenesis in domestic animals has been investigated and there is little information. The expression of CD9 and SYCP3 mRNA were examined using RT‐PCR for immature bull testes (Fig. 4). CD9 expression at 4 weeks in immature bull testes showed lower levels than day 2–5 mouse testes. This difference may be caused by species specificity. Spermatogonial stem cell transplantation can potentially contribute to studies of gene expression during spermatogenesis and then used in domestic animals as a means to preserve a male's germ cell line, increase genetic progress in domestic animals, and generate transgenic animals. Bovine spermatogonial stem cells have been shown to be capable of colonizing recipient mouse seminiferous tubules; however, no studies have shown them undergoing differentiation into spermatozoa. ¹⁹ , ²² , ⁷²

Figure 4.

Expression of CD9 and SYCP3 mRNA was examined using reverse transcriptase‐polymerase chain reaction for immature bull testes. The values are means of the ratio of CD9 and SYCP3 mRNA mRNA to β‐actin mRNA.

In recent years, in vitro culture of bovine germ cells without feeder cells has been demonstrated. Lee et al. ²¹ used a calcium–alginate encapsulation method and demonstrated differentiation of bovine gonocytes to haploid cells; however, spermatogonial stem cell maintenance was not evaluated. Izadyar et al. ⁷³ used a gel matrix to support the proliferation and differentiation of bovine spermatogonia in vitro; however, analysis of spermatogonial stem cell survival was limited because of the lack of a labeling method for the donor cells. Previously Oatley et al. demonstrated the survival and proliferation of bovine spermatogonial stem cells over a 1‐week period using a testis tissue explant culture system. ⁷² A system that allows for the culture of a single‐cell suspension of bovine spermatogonial stem cells would be more beneficial for long‐term survival and genetic modification. Culture and cryopreservation methods for spermatogonial stem cells could be used to immortalize a male's genetic line through the germ cells as a result of the stem cells’ ability to self‐renew. The efficacy of this approach with spermatogonial stem cells of domestic animals has not been clearly demonstrated. An in vitro system that supports spermatogonial stem cell survival and proliferation is of benefit for the enhancement of stem cell numbers and genetic modification. Based on previous studies in rodents, the culture of bovine spermatogonial stem cells as a single‐cell suspension must be carried out on a feeder cell monolayer. The round cell morphology of colonized bovine cells in mouse seminiferous tubules was consistent with previous reports of other cross‐species testicular transplantations. ¹⁹ , ²² , ²⁴ , ²⁵ , ⁷² Their observation was in contrast to previous reports of somatic cell colonization in a recipient testis. ⁵ , ⁷⁴ , ⁷⁵ The donor cells were recovered from bulls 4–8 weeks of age, a developmental stage in which gonocytes or spermatogonial stem cells are the only cell type present within the testis. Therefore, it is highly likely that any germ cell colonization in recipient mouse testes was undifferentiated spermatogonial stem cells rather than differentiated germ cell types. It appears that spermatogonial stem cells from many species are quite robust and capable of survival during cryopreservation in relatively simple conditions.

The culture of bovine germ cells on bovine embryonic fibroblast (BEF) feeder cells for 1 week resulted in a significant increase in round cell colonies following transplantation compared to frozen‐thawed germ cells transplanted prior to culture. ³⁷ It was strongly indicated that bovine spermatogonial stem cells had been surviving and proliferating over a 1‐week period. Mouse spermatogonial stem cells have been shown to decrease during the first 7 days of culture on feeder cells. ³⁷ However, the addition of GDNF to the medium enhanced spermatogonial stem cell numbers in these cultures. It has been demonstrated that GDNF is a factor for spermatogonial stem cell maintenance. ³⁷ A significant decrease in bovine colony numbers in recipient mouse testes arising from 2 weeks cultured bovine germ cells has been observed, and indicates that spermatogonial stem cell death or stem cell differentiation to committed spermatogonia occurred over a 2‐week period, thereby, decreasing the percentage of stem cells within the overall germ cell population. Currently, the means of action that GDNF has on spermatogonial stem cell maintenance is unknown. Whether it inhibits stem cell differentiation or promotes stem cell proliferation needs through discussion. If stem cell differentiation was occurring over a 2‐week period, higher stem cell populations may have been maintained compared with cultures without added exogenous GDNF. The maintenance of these cells may have been through inhibition of stem cell differentiation as proposed by Nagano et al. ³⁷ or by inhibition of cell death. If activation of GDNF on stem cells works through inhibiting differentiation, then stem cell numbers would increase because of auto self renewal. An effect of GDNF on spermatogonial stem cell maintenance was demonstrated using this culture system. A BEF feeder cell co‐culture system was developed that supports the maintenance of bovine spermatogonial stem cells. The BEF co‐culture system reported has the potential to be used as a means for accomplishing stable genetic modification and for investigating factors regulating the biological activity of spermatogonial stem cells.

Development of in vitro culture and cryopreservation techniques for livestock spermatogonial stem cells are necessary for the application of spermatogonial stem cell transplantation. These technologies can potentially be used as a reproductive tool to enhance genetic progress and generate transgenic animals using the male germ line.

CONCLUSION

RECENTLY, KNOWLEDGE AND information of male germ cell biology has been advanced. The knowledge, as well as the morphology of spermatogenesis, the gene expression of spermatogonial stem cells and culture methods developed, holds great promise in treating male infertility and perhaps in germline gene therapy. The next advances will be in domestic animal spermatogonial stem cell culture and in vitro differentiation of the cultured stem cells. Germ cell transplantation is a powerful tool to explore basic biological aspects of male germ line stem cells and testis function of domestic animals. Practical applications include the introduction of genetic modifications into the germ line of domestic animals, and the preservation of fertility from valuable individuals. Transplantation of germ cells or testis tissue will continue to significantly enhance our understanding of testis function and our ability to control and preserve male reproductive biology.