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Gamete Biology |
Department of Obstetrics and Gynecology,4 Baylor College of Medicine, Houston, Texas 77030
Department of Molecular and Human Genetics,5 Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, gametogenesis, sperm, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The germ cell-deficient (gcd) mutation is a recessive, transgenic insertional mutation in which a drastic depletion of PGCs occurs in the developing genital ridge, resulting in infertility in both male and female adults [11]. We have recently demonstrated that the mutation is caused by a deletion functionally eliminating Vrk2 (vaccinia virus-related protein kinase 2) and Pog (proliferation of germ cells). Targeted deletion of Pog mirrors the gcd phenotype in that the number of PGCs in 10.5–13.5 dpc Pog-/- embryos is lower than that in normal control embryos [12]. Furthermore, similar to gcd/gcd mice, 4- to 6-wk-old POG-deficient mice have virtually no developing follicles in females and no spermatogenesis in the majority of seminiferous tubules in males. We have also shown that this is caused by abnormal proliferation of PGCs rather than by defective migration [12].
A number of genes have been found to be important for various aspects of PGC development. Bmp2, Bmp4, Bmp8b, and genes involved in transducing the BMP signals (e.g., Smad2 and Smad5) into the cell are important for the generation of PGCs [13–15]. Fancc, Tial1(TIAR), Kit, and Kitl are important for the proliferation, survival, or migration of PGCs [16–20]. Whereas Fancc-/-, Bmp4+/-, Slpan/Slpan (Kitl mutant), and Slcon/Slcon (Kitl mutant) mice all have various degrees of PGC deficiency during the embryonic stage, the adult males are fertile [13, 19, 21]. This also indicates that suboptimal numbers of spermatogonia have the ability to re-establish the germ stem cell pool and to maintain normal spermatogenesis. The gcd/gcd males are reported to be infertile, although PGCs do exist at 13.5 dpc [11]. When we looked at the spermatogenesis in Pog-/- mice at different ages, we noticed a late establishment of the germ cell population in more than 90% of the seminiferous tubules and a subsequent gain of fertility in most of the Pog-/- mice at 12 wk of age. Thus, although Pog-/- and gcd/gcd females show the same phenotype in terms of infertility and ovarian morphology, Pog-/- and gcd/gcd males have different reproductive potentials. Further analysis suggests that the difference may be the result of much more severe germ cell deficiency at 13.5 dpc and 1 day postpartum (dpp) in gcd/gcd males. Our data suggest that Pog is necessary for PGC development but is not necessary for the subsequent proliferation of spermatogonia. The Pog-/- male, which is infertile at first and then later gains fertility, represents a unique model for studying the stem cell biology of germ cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experiments on animals were conducted in accordance with the National Research Council publication Guide for Care and Use of Laboratory Animals. Heterozygous gcd/+ mice were maintained on a C57BL/6 background. Because most of the gcd homozygotes embryos die in a pure C57BL/6 background (unpublished observations), homozygous gcd/gcd mice were generated by intercrossing (FVB x C57BL/6)F1 gcd/+ heterozygotes. Generation of Pog knockout mice, genotyping the Pog targeted allele, and genotyping of gcd/gcd mice were performed as previously described [12]. Pog-/- mice were obtained by intercrossing (FVB x 29Sv)F1 Pog+/- heterozygotes. Female gcd/+ of (FVB x C57BL/6)F1 were mated with male Pog+/- (FVB x 129Sv)F1 to obtain Pog null embryos harboring the gcd allele and targeted Pog allele (compound heterozygotes). The morning that a coitum plug was found was counted as 0.5 dpc. The day after pups were born was counted as 1 dpp.
Testis Histology
Tissues were fixed in 4% paraformaldehyde or Bouin solution overnight at 4°C, dehydrated, and embedded in Paraplast X-tra (Fisher Scientific, Pittsburgh, PA). Sections (thickness, 8 µm) were stained with hematoxylin/eosin (H&E) or periodic acid-Schiff reagent.
Epididymal Sperm Counts and Fertility Testing
One testis was dissected and weighed without fat tissue or the epididymis. Both the caput and caudal epididymis from one testis were then dissected and minced in a 24-well plate in PBS (free of calcium and magnesium). The volume of the solution was adjusted to 1 ml with PBS, and the minced tissue was incubated at 37°C for 20 min to allow the sperm to swim out of the epididymis. The number of sperm per 1 ml was counted with hemocytometry. For fertility testing, the test male was caged with three normal, mature females for the time indicated. Copulation was checked by the presence of coitum plug.
Antibody Staining of Germ Cells in 13.5-dpc Embryos
Tissue sections were dewaxed, rehydrated, and boiled for 20 min in 50 mmol/L of Tris-HCl (pH 9.5). The sections were blocked with 10% normal serum in PBS and incubated with anti-POU5f1 (OCT4) antibody (1:200 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4°C. Vector Elite ABC kit (Vector Laboratories, Burlingame, CA) was used to detect the POU5f1-positive germ cells.
Seminiferous Tubules and Germ Cell Number Analysis
Numbers of seminiferous tubules with or without germ cells were counted on the printed photograph of the H&E-stained testes sections. To compare the seminiferous tubule diameter, the H&E-stained testes sections from mutants and controls were photographed and printed with the same magnification, and the diameters of the round tubules (cross-section of the tubule) were measured. To count the germ cell number, the germ cells of 13.5-dpc embryos were stained with anti-POU5f1 antibody as described above. Two normal controls, three Pog-/- embryos, and two gcd/gcd embryos were analyzed. The POU5f1-positive germ cells were counted from indicated tubules out of multiple sections at least three sections apart. Only cross-sections of the tubules were included in the analysis. For 1-dpp testis, germ cells were recognized by their large, round morphology after H&E staining. Three pups of each genotype were analyzed. The number of germ cells was expressed as cells per cross-section of the tubules.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When the spermatogenesis of Pog-/- mice was examined at different ages, it was noticed that the germ cells populate the seminiferous epithelium gradually with increasing age (Fig. 1). We checked four to six Pog-/- mice by H&E staining of testes sections at 4, 6, 9, 12, 16, and 22 wk of age. In 4-wk-old Pog-/- testes, most of the seminiferous tubules either did not have germ cells at all (Sertoli cells only) or had a few primary spermatocytes as the most advanced cell type (Fig. 1, A and B). Three 4-wk-old Pog-/- testes were analyzed quantitatively, and each contained 62%, 64%, and 42% tubular sections without germ cells or had a few germ cells but without obvious spermatogenesis. At 9 wk, significantly more tubules had full spermatogenesis, and the percentage of tubules with no germ cells decreased to less than 10% (Fig. 1, C and D). At 12 wk, all the cross-sections of the seminiferous tubules showed full spermatogenesis, with the exception of fewer than 10 tubules on each testis section that were devoid of germ cells. In testes of 16- and 22-wk-old males, we still noticed similar numbers of tubules devoid of germ cells (Fig. 1, E and F). These "empty" tubules were usually found in clusters and may represent the areas that originally had no stem cells. It is probable that these tubules might remain unpopulated during the rest of the lifetime.
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In terms of spermatogenesis, after 12 wk, almost all the seminiferous tubules manifested an "all or nothing" state, indicating that the process of population happened before that time. Consistent with this, tubules with a whole germ cell layer missing were readily seen in 6-wk-old Pog-/- testis (Fig. 1G), whereas they were not seen after 12 wk. The missing of a wave of spermatogenesis could be the result of a biased, asymmetric division of germ stem cells. This would have the effect of generating more self-renewing stem cells to establish the stem cell pool, resulting in less or even no differentiating spermatogonia for another wave of spermatogenesis. The absence of such tubules in testes after 12 wk again suggested that the process of establishing the germ stem cell pool in Pog-/- mice was basically finished before 12 wk.
Although the spermatogenesis in the populated seminiferous epithelium looked qualitatively normal, in that four waves of spermatogenesis were found in each cross-section of the tubules, it was not quantitatively normal. Compared with age-matched normal controls, Pog-/- testes had a lower ratio of spermatocytes to round spermatids, although they had a normal ratio of 3N cells (majority proliferating spermatogonia; unpublished observations). Furthermore, compared with normal controls, Pog-/- testes had a smaller number of tubules (data not shown), smaller tubular diameter, and smaller tubular lumens (Fig. 1, D, I, and J). In some Pog-/- testes, sloughing of immature germ cells could be seen in the tubular lumen (Fig. 1H).
Pog-/- Males Become Fertile after 12 Wk
We used testis weight and epididymal sperm number as indicators to further examine spermatogenesis of Pog-/- males. At 6–7 wk, when normal littermates reach reproductive maturity, Pog-/- males had very small testis, with no sperm in the epididymis. At 9 wk, with population of the seminiferous epithelium started, we saw a small but significant increase in testis weight (Fig. 2A and Table 1). However, the testes from Pog-/- mice never reached the size of the testes from the age-matched, normal controls. Even at 22 wk, most of the mutant testes were less than half the weight of normal controls (Fig. 2A). This may be the result of the fewer seminiferous tubules and smaller tubular diameter that we observed in Pog-/- mice. The body weight of the Pog-/- mice did not show significant difference compared with those of the normal littermates (data not shown). The number of spermatozoa in the epididymis increased with the population of the seminiferous epithelium and the resumption of the spermatogenesis. At 6–7 wk, most Pog-/- mice had no spermatozoa in the epididymis. At 9 wk, Pog-/- mice had approximately 4% the number of epididymal sperm compared to normal controls. By 12 wk, the number increased to 10%, and at 22 wk, some males attained approximately 50% normal numbers (Fig. 2B). A large variation was observed in the testes weight and epididymal sperm numbers between Pog-/- individuals, although the testis size correlated well with the sperm number. In the mutants, larger testes had more epididymal sperm. Although most of the Pog-/- mice showed population of the seminiferous tubules with germ cells, we did find one male with extremely small testes and low sperm counts, even at 22 wk of age.
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We tested the fertility of Pog-/- males at 12 wk of age by mating each of two Pog-/- males with three normal C57BL/6 females. Both were found to be fertile, giving normal litter sizes. Twelve pups from two litters were genotyped, and all of them carried the targeted allele, confirming the parental male homozygous genotype. This is in agreement with the case of Trp53-/-;Kit(WV) mice, in which 10% of normal sperm (assuming the average normal sperm number in each epididymis is 
2 x 107) was shown to be sufficient to maintain normal fertility [22].
Spermatogenesis and Fertility of gcd/gcd Males
To our knowledge, population of the seminiferous epithelium by germ cells and the gain of fertility in older Pog-/- males have not been systematically analyzed in older gcd males. This prompted us to reinvestigate the spermatogenesis in gcd/gcd males over a similar time course. Again, at least four gcd/gcd males were checked at 4, 9, 12, 16, and 22 wk. In gcd/gcd males, the onset of spermatogenesis was similarly delayed. At 4–6 wk, none of the seminiferous tubules in gcd/gcd testes showed full spermatogenesis (Fig. 3A). At 9 wk, some tubules with complete spermatogenesis could be seen. However, the percentage of tubules with full spermatogenesis did not increase as much as in Pog-/- mice. Even at 16 and 22 wk, 40–70% of tubules were still devoid of germ cells (Fig. 3, B and C). Hyperplasia of the interstitial somatic cells was evident, especially in those testes with a high percentage of tubules devoid of germ cells (Fig. 3B). More striking differences between gcd/gcd and Pog-/- mice were found in the testes weight and the epididymal sperm counts. The testes weight was significantly lower in gcd/gcd mice than in age-matched Pog-/- mice (Fig. 2A). The gcd/gcd males had smaller testes than Pog-/- males at all time points checked. At 16 and 22 wk, most gcd/gcd mice had less epididymal sperm than 9-wk-old Pog-/- mice (Fig. 2B). In some of the 16- and 22-wk-old males, no sperm could be seen in the epididymis at all (Fig. 3D). Two 16-wk-old gcd/gcd males were each mated to three normal females, and no pregnancies occurred after 3 wk of mating. This is consistent with the very low sperm number seen in gcd/gcd mice. At 22 wk, only 2 of 10 gcd/gcd mice were found to have more than 2 x 106 sperm in the epididymis and to show population of the germ cells in a majority of the seminiferous tubules (Fig. 3E). This is consistent with the previous report that 13 matings of gcd/gcd males with normal females produced only one pregnancy [11]. In our breeding program, we have identified only one fertile gcd/gcd male (unpublished observations).
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gcd/gcd Males Have Fewer Germ Cells than Pog-/- Males Before the Onset of Spermatogenesis
Prospermatogonia in the mouse testis resume mitosis after birth. To determine whether the difference in spermatogenesis in gcd/gcd and Pog-/- mice was the result of a defect in gcd/gcd spermatogonial proliferation in adulthood or of a more severe germ cell deficiency before the onset of spermatogenesis, germ cell numbers were compared at 1 dpp and 13.5 dpc. The results revealed that at both 1 dpp and 13.5 dpc, gcd/gcd males had a much more severe germ cell deficiency than Pog-/- males (Tables 2 and 3 and Fig. 4). This was evident in both the average number of germ cells per cross-section of the tubules and the percentage of cross-section of tubules without germ cells. At both time points, gcd/gcd males had a lower percentage of germ cells relative to their respective controls than Pog-/- males (last row of Tables 2 and 3). The more dramatic difference resided in the fact that at both time points, gcd/gcd males had a higher percentage of tubules with no germ cells. Thus, gcd/gcd males showed a much more severe germ cell deficiency before the onset of spermatogenesis.
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At both time points, we noticed that in the mutants (gcd/gcd and Pog-/-), the diameter of the seminiferous tubules was smaller than that in normal controls (Table 4), which is consistent with our observations that the seminiferous tubules of adult gcd/gcd and Pog-/- mice also had a smaller diameter than in their normal littermates.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have shown recently that Pog is the gene underlying the gcd mutation, being essential for the efficient proliferation of PGCs during embryonic stages [12]. In the present study, we show that Pog-/- males, although deficient in germ cells before the onset of spermatogenesis, finally become fertile because of relatively late population of the seminiferous tubules by germ cells and the resumption of spermatogenesis after 3–4 mo of age. This suggests that Pog is important for the proliferation of PGCs at the embryonic stage but is not important for the early proliferation of spermatogonia in the adult. Our results suggest that the inefficient number of germ cells before the onset of spermatogenesis may be the main reason for late onset of spermatogenesis in Pog-/- mice. Because spermatogenesis is a process tightly regulated by a series of hormones, such as gonadotropins and testosterone, and because gcd/gcd males are reported to have elevated FSH levels [23], whether Pog-/- males have hormone imbalance and the nature and effects of hormone imbalance on this process need to be investigated.
We noticed that at 13.5 dpc, 1 dpp, and adulthood, mutants (gcd/gcd and Pog-/-) had smaller seminiferous tubule diameters than in their normal littermates. Although the difference was statistically significant, the seminiferous tubule diameter of mutant testes decreased only by approximately 12%–20% relative to normal testes. We also noticed that in both the adult gcd/gcd and Pog-/- testes, tubules with full spermatogenesis had larger diameters than tubules without germ cells or with poor spermatogenesis. Considering the report that busulfan-treated, germ cell-deficient rat gonads had smaller tubule diameter [24], it is difficult to distinguish the direct effect of deletion of Pog on Sertoli cells from the secondary effect of germ cell deficiency. In agreement with previous reports [11], our data suggest that the Sertoli cells in gcd/gcd and Pog-/- mice were functionally normal, because they could support qualitatively normal spermatogenesis.
Population of the seminiferous tubules by germ cells was not as complete in gcd/gcd mice as in Pog-/- adult mice. This may be a direct reflection of the fewer germ cells seen in gcd homozygotes compared to POG-deficient males rather than a reduced proliferation of the gcd/gcd spermatogonia. The fact that a few gcd/gcd males at 16 and 22 wk had the same degree of germ cell population as seen in Pog-/- males also supports this view. Furthermore, gcd/gcd seminiferous tubules manifested an "all or nothing" phenotype in terms of spermatogenesis. Clustering of tubules devoid of germ cells suggested that the residual gcd/gcd spermatogonia, although in a smaller number, could still establish a focal stem cell pool and maintain qualitatively normal spermatogenesis in those tubules that originally had spermatogonia.
Why gcd/gcd males have a much more severe germ cell deficiency than Pog-/- males before the onset of spermatogenesis is not clear. Two explanations for this difference are possible. First, two genes (Vrk2 and Pog) are deleted in the gcd mouse. Vrk2 is reported to have high expression in actively proliferating cells, and it is expressed in 10.5 dpc embryos [25]. It is possible that although deletion of Pog is enough to cause germ cell deficiency, deletion of Vrk2 has an added deleterious effect on germ cell proliferation or survival. Second, the gcd/gcd mice and Pog-/- mice were on different genetic backgrounds. The gcd/gcd mice were obtained by intercrossing (FVB x C57/BL6)F1 gcd/+ heterozygotes, whereas Pog-/- mice were obtained by intercrossing (FVB x 129)F1 Pog/+ heterozygotes. It is possible that different background accounts for the different severity of germ cell deficiency in gcd/gcd and Pog-/- mice. We noticed that on a majority C57BL/6 background, both the Pog-/- and the gcd/gcd homozygote pups were severely underrepresented, indicating embryonic lethality, whereas on the FVB background, they were found in the expected mendelian ratios (unpublished observations). This means that the genetic background does affect the outcome of the Pog deletion. Thus, although we cannot exclude the possibility that Vrk2 is directly involved, we favor the hypothesis that the differences are more likely caused by genetic background. Targeting of Vrk2 and/or comparison of the germ cell deficiency between Pog-/- and gcd/gcd mice on identical genetic backgrounds will be needed to address this issue.
Repopulation of the germ cells has been studied with busulfan- or radiation-treated animals [6–8] and with germ cell-transplanted animals [26–29]. The germ cell deficiency before the onset of spermatogenesis and the re-establishment of the germ stem cell pool after birth in Pog-/- males makes the POG-deficient mouse a unique model for the study of germ stem cell proliferation. Introducing visible markers such as a Pou5f1 (Oct-4) promoter-driven green fluorescent protein (GFP) [30] or ß-galactosidase into the background to specifically label the germ cells would enhance its value as a model system in this respect.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Colin E. Bishop, Department of Obstetrics & Gynecology, Baylor College of Medicine, 6550 Fannin Street (#880), Houston, TX 77030. FAX: 713 798 5074; bishop{at}bcm.tmc.edu
3 Current address: Beijing Institute of Biotechnology, Beijing, China, 100071
Received: 13 December 2002.
First decision: 6 January 2003.
Accepted: 3 February 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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