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Regular Article |
a Genetic Diseases Research Branch, National Human Genome Research Institute,
b Veterinary Resources Program, Office of Director, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, phosphatases, Sertoli cells, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Little is known regarding the mechanisms involved in the translocation. It has been hypothesized that the adherens junctions, composed of N-cadherin and its cytosolic binding proteins, the catenins [2], may be internalized by endocytosis and recycled to the new site of adhesion during translocation of germ cells [3]. In support of the above hypothesis, vesicles that carry intact adherens junctions in the seminiferous tubules of mink testes have been visualized by electron microscopy [4].
The role of phosphoinositides as regulators of membrane traffic is well established [5, 6]. Phosphoinositides phosphorylated at position 5 of the inositol ring, PtdIns(4,5)P2 and PtdIns(3,4,5)P3, have been implicated in regulating secretion from neuroendocrine cells [7], clathrin-mediated endocytosis [8], and phagocytosis [9]. PtdIns(3)P and other higher phosphorylated products of PtdIns 3-kinase regulate endocytosis and endosome-endosome fusion [10]. Phosphoinositides also function in a variety of signal transduction pathways, ranging from cell growth and differentiation to apoptosis and cytoskeletal rearrangements [11]. The effects of D5 phosphoinositides are believed to be silenced by hydrolysis of the D5 phosphate through action of a 5-phosphatase such as Inpp5b. Inpp5b is ubiquitously expressed in mouse tissues [12], and the protein displays broad substrate specificity in vitro by acting both on lipid substrates PtdIns(4,5)P2 and PtdIns(3,4,5)P3 and on soluble inositol polyphosphates Ins(1,4,5)P3 and Ins(1,3,4,5)P4 [13].
Here, we present detailed phenotypic analyses of mice deficient in Inpp5b. We show that loss of function of Inpp5b results in the appearance of N-cadherin- and ß-catenin-containing vacuoles in the cytosol of Sertoli cells of the testis. The vacuoles in the Inpp5b-/- animals are coated with actin and contain proteins normally concentrated at the Sertoli cell surface, suggesting that the vacuoles may represent swollen endosomal structures formed due to an early block in apical endocytosis before uncoating. We propose that Inpp5b may function in the endocytosis and recycling of plasma membrane in the testes, thereby affecting cell adhesion to neighboring cells and resulting in concomitant, premature release of germ cells from the mutant seminiferous epithelium.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generation of inbred 129S6/SvEvTac.Inpp5b-/- mice has been described previously [12]. For assessing fertility, mutant males were mated with at least one fertile female over a period ranging from 8 wk to 9 mo, and litter numbers and sizes were scored. All animal experiments were conducted with the approval of the National Human Genome Research Institute's Animal Care and Use Committee according to National Institutes of Health Guidelines on Care and Use of Laboratory Animals.
Enzyme Assays
Testes of 3-mo-old wild-type and Inpp5b-/- animals were dissected and homogenized in homogenization buffer containing 0.25 M sucrose, 10 mM Hepes (pH 7.4), 0.1 mM EGTA, and 0.1 M dithiothreitol containing 1 µg/ml of leupeptin and 1 µg/ml of aprotinin. Before enzyme assays, 20 µg of protein were analyzed by Western blotting using anti-Inpp5b antibody (1:70 [v/v]) to verify the presence of Inpp5b in the samples as described elsewhere [14]. PtdIns(4,5)P2 5-phosphatase enzyme assays were performed in duplicates using [3H]PtdIns(4,5)P2 as a substrate on 20 µg of testis protein essentially as described previously [15], except that the assays were performed with the 2.5:1 (v/v) ratio of cetyltriethylammonium bromide (CTAB) detergent to substrate in the reaction mixture. Ins(1,4,5)P3 5-phosphatase assays were performed in duplicates as described previously [16].
Histology and Immunohistochemistry
Tissues fixed in Bouin fixative were cut in half and embedded in epon, and 2-µm cross-sections were stained with hematoxylin-eosin (H&E) or toluidine blue. For each time point, at least three animals were killed. Tissues to be stained with periodic acid-Schiff (PAS), PAS + diastase (detection of glycogen), Sudan black, and Oil Red O (detection of lipids) were snap-frozen by dropping in liquid nitrogen and embedded in O.C.T. (Ted Pella, Inc., Redding, CA), and 6-µm sections were then stained according to routine protocols. For immunohistochemistry with antibodies to N-cadherin (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), ß-catenin (1:250; Zymed Laboratories, San Francisco, CA), actin (1:1000; Santa Cruz Biotechnology), transferrin (Tf; 1:50; Accurate Chemical & Scientific Corporation, Westbury, NY), Tf receptor (TfR; 1:50; Santa Cruz Biotechnology), and clusterin (1:500; Upstate Biotechnology, Inc., Lake Placid, NY), 6-µm tissue sections were formalin-fixed, paraffin-embedded, and subjected to antigen retrieval by boiling for 10 min in 0.01 M citrate buffer. Immunohistochemistry was performed using the ABC kit (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA) according to the manufacturer's protocols. The specificity of the antibodies used was confirmed by Western blot analysis in which all produced a single band in the testis lysates. Specific staining was further evaluated by proper controls in immunohistochemistry (e.g., omitting primary antibody, secondary antibody, or both from the processed slides). TUNEL assays were performed on paraffin-embedded, 6-µm sections as described previously [17]. Two animals were killed at each time point. For each testis, the number of TUNEL-positive nuclei in 100 magnification fields (400x) was determined.
Electron Microscopy
Testes were perfused with 2.5% (w/v) glutaraldehyde and 2% (w/v) formaldehyde in 0.1 M Sorenson buffer by the vascular route. Perfused testes were postfixed overnight in the same fixative, rinsed, and fixed in 2% (w/v) OsO4 for 1 h. Testes were dehydrated and embedded in epon. Thick sections (1 µm) were stained with toluidine blue and examined by light microscopy to determine which blocks were to be thin-sectioned and subsequently examined by electron microscopy.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generation of mice deficient in Inpp5b has been described previously [12]. Homozygous mutant female and male mice were indistinguishable in growth and development from their normal littermates. However, the mutant males showed reduced fertility, with only one litter of three pups born to 1 of 17 males whose fertility was extensively tested (Table 1), whereas Inpp5b-/- females were fertile. All Inpp5b-/- males produced vaginal plugs. No gross abnormalities were observed in tissue sections from all major organs except the testis. Seminal vesicle, epididymis, and testis weights (measured as a percentage of body weight ± SD) were normal at 14 wk in mutant mice, indicating normal levels of circulating androgens (testis: wild-type, n = 8, 0.37% ± 0.01%; knock-out, n = 8, 0.34% ± 0.03%; epididymis: wild-type, n = 8, 0.13% ± 0.01%; knock-out, n = 8, 0.12% ± 0.02%; seminal vesicles: wild-type, n = 6, 0.35% ± 0.02%; knock-out, n = 6, 0.34% ± 0.04%). We saw no rescue of the phenotype after breeding Inpp5b-/- females with wild-type 129S6 males to introduce a Y chromosome different from the one originally present in the targeted embryonic stem (ES) cells, thus ruling out the possibility of an abnormal Y chromosome in the ES cells contributing to infertility.
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We verified that the Inpp5b-/- mice had reduced phosphoinositide 5-phosphatase activity in testes homogenates by assaying at a low CTAB:substrate ratio of 2.5:1 [12]. Dephosphorylation of [3H]PtdIns(4,5)P2 5-phosphate to PtdIns(4)P in the mutant animals was significantly reduced to 46% of control (P = 0.014) (Fig. 1A), with the residual activity most likely representing another 5-phosphatase present in the testes. This residual activity, however, is not the Ocrl1 enzyme, because very little phosphoinositide 5-phosphatase activity was ascribable to Ocrl1 in either the control or the mutant testes homogenates as determined by assaying at a high CTAB:substrate ratio of 10:1 (data not shown). We have previously shown that, in mouse fibroblasts, Ocrl1 is active at a high (10:1) but inactive at a low (2.5:1) CTAB:substrate ratio [12]. This suggests that testes may be particularly vulnerable to a deficiency of Inpp5b because they lack compensation from Ocrl1. We observed only a modest reduction, to 85% of control, in the Ins(1,4,5)P3 5-phosphatase activity in the mutant testes (Fig. 1B).
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Reversible Disruption of Spermatogenesis in Mice Deficient in Inpp5b
Spermatogenesis was disrupted at meiosis I with concomitant appearance of vacuoles in the mutant seminiferous tubules. The abnormalities followed a striking biphasic course (Fig. 2). Vacuoles first appeared in the mutant testes at Day 13 (Fig. 2B), the age at which differentiating germ cells entered meiosis I. At Day 21, mutant testes showed a loss of meiotic germ cells and massive vacuolization in the seminiferous epithelium (Fig. 2D). We rarely saw spermatids in the mutant testes, and the remaining meiotic spermatocytes were often located in the lumen detached from the epithelium. The vacuoles did not contain substantial amounts of lipid, glycoprotein, or glycogen based on their lack of staining with routine histological stains used to detect these molecules. However, H&E staining showed the presence of scant proteinaceous material inside the vacuoles at 14 days, but not at later time points, in the mutant testes (data not shown). Remarkably, at 6 wk of age, the mutant testes histology began to recover, and substantial numbers of germ cells, including sperm, became evident (Fig. 2F). Spermatogenesis appeared to be normal in the mutant testes between 2 and 5 mo, but Inpp5b-/- males remained compromised in their fertility. With further aging, mutant testes began to display progressive cell drop-out and germ cell loss (Fig. 2H). By 1 yr of age, most tubules from Inpp5b-/- animals had a severe disruption in spermatogenesis, characterized by complete loss of spermatocytes and spermatids but relative preservation of Sertoli cells and spermatogonia.
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Germ Cell Fate in the Testis of Inpp5b-Deficient Animals
Testicular maturity is achieved in normal testis, at least in part, through an early apoptotic wave, when excess spermatocytes are removed from the epithelium between 2 and 5 wk. This programmed cell death is required to maintain the correct ratio of Sertoli and germ cells that is essential for successful spermatogenesis [18]. We therefore used the TUNEL assay to determine if an abnormally high level of apoptosis is the cause of the decrease in spermatocytes and spermatids in the young mutant animals (Table 2). At 2 wk, fewer apoptotic nuclei were observed in the testes sections of mutant animals compared to control animals, and at 4 wk, the number of apoptotic nuclei was similar between mutant and control animals. In contrast, we saw significantly more apoptotic nuclei in the testis sections of adult, 4- to 6-mo-old knock-out animals compared to the control animals. As an alternative to excessive apoptosis, defective cell adhesion between Sertoli cells and germ cells could cause the cellular loss during the first spermatogenic wave in the mutant testis. In that case, immature germ cells would be lost into the tubular lumen and transported to the epididymis. Indeed, we saw an excess of round germ cells in the lumen of the caudae epididymides of 4-wk-old mutant mice (Fig. 3). At 6 mo, mutant epididymides displayed normal histology, with no evidence of immature germ cells in the lumen (data not shown).
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Adherens Junctions Are Trapped into Swollen Endosomes in the Inpp5b-/- Testes
The ultrastructure of mutant testes appeared to be normal during electron microscopic examination at the ages of 16 days (data not shown), 22 wk (Fig. 4), and 1 yr (data not shown), except for the presence of large vacuoles located in the cytoplasm of the Sertoli cells (Fig. 4B, arrow), which in some cases filled the whole Sertoli cytosol. The vacuoles were surrounded by membrane and occasionally had membranous material and multivesicular body-like structures inside. Budding and/or fusion of small vesicular structures from and/or to vacuoles was evident. Many vacuoles were surrounded by hexagonally packed filaments (Fig. 4, C and D, arrowheads), with elongated sacs of endoplasmic reticulum located beneath them. This morphological structure is characteristic of adherens junctions, which serve as one of the main adhesion complexes that attach meiotic germ cells to the seminiferous epithelium [2].
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We confirmed the presence of adherens junctions on these vacuoles by immunostaining testes sections from 14- and 21-day-old control and mutant animals with antibodies against N-cadherin (the major cadherin in the normal mouse testis [19]), ß-catenin, and actin. We chose these two time points to analyze vacuoles when they first appeared at 14 days, when mutant testes histology was otherwise fairly normal, and 1 wk later to observe any changes in the expression of proteins localized to intercellular junctions as they are redistributed in the testes on aging of the animals [20]. In control testes from both 14-day-old (Fig. 5) and 21-day-old (data not shown) mice, N-cadherin (Fig. 5A), ß-catenin (Fig. 5D), and actin (Fig. 5G) immunoreactivities were present between spermatocyte and Sertoli cell contacts, on Sertoli and germ cell membranes and in their cytosol, and on interstitial cells. These results are compatible with those of a previous report [20], except that in our experiments, N-cadherin immunoreactivity was also localized close to the basement membrane, which most likely represents staining of adherens junctions between neighboring Sertoli cells. This discrepancy may be due to differences in the antibodies and fixation methods used in this and the previous study. In contrast, mutant testes from 14-day-old animals showed N-cadherin (Fig. 5B), ß-catenin (Fig. 5E), and actin (Fig. 5H) immunoreactivities around large vacuoles in addition to staining compatible with what was observed in control sections with these antibodies. All antibodies also stained proteinaceous material inside the vacuoles in 14-day-old mutant animals. This staining most likely corresponds to the H&E-positive material that was evident inside the vacuoles at 14 days but not at older ages. At 21 days, N-cadherin (Fig. 5C), ß-catenin (Fig. 5F), and actin (Fig. 5I) staining was concentrated around these abnormal structures in the mutant testes, with reduced staining of Sertoli cell cytosol. At 21 days, most of the spermatocytes were lost from the mutant testes; however, the remaining spermatocytes showed normal staining between cellular contacts and in the cytosol with all antibodies. Of note, ß-catenin staining was punctate and clustered on specific membrane domains in the mutant testes at 21 days (arrow in Fig. 5F) but not at 14 days, indicating the presence of intact adherens junctions at these sites.
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Accumulation of Both Apical and Basolateral Resident Proteins in Vacuolar Structures of Inpp5b-/- Mice
In the testis, adherens junctions reside on the apical membranes adjacent to tight junctions [4]. Our findings suggested that loss of function of Inpp5b results in disturbances of adherens junction recycling and, therefore, of apical endocytosis. We wanted to test this hypothesis using additional protein markers and to determine whether basolateral endocytosis was affected in Inpp5b-/- mutant animals as well. To this aim, we stained mouse testes sections with antibodies against differentially targeted proteins, including clusterin (SGP2, ApoJ), Tf, and TfR. Clusterin is exclusively targeted to the apical membrane in Sertoli cells by an as-yet-uncharacterized mechanism [21]. In the control 14-day (Fig. 6A) and 21-day (data not shown) testes, clusterin immunoreactivity was present in the Sertoli cell cytosol and as punctate spots between Sertoli and spermatocyte contacts, as described previously [22]. In mutant testes, punctate clusterin immunoreactivity was present around enlarged vacuoles at 14 days as well as in the aggregates inside the endosomes (Fig. 6B). At 21 days, nearly all clusterin staining was associated with vacuoles in the mutant testes (Fig. 6C), with reduced staining of the Sertoli cell cytosol. Because clusterin expression is up-regulated at approximately 3 wk [23], staining was, as expected, more intense at 21 days compared to that at 14 days in both control and mutant testes.
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Recycling of Tf and TfR from the basal membrane is well characterized in Sertoli cells [24]. Sertoli cells also newly synthesize Tf and target it apically in vivo [25]. In the 14-day (Fig. 6D) and 21-day (data not shown) control testes, Tf immunoreactivity was present on meiotic spermatocytes and in the interstitial space. In the mutant testes, Tf immunoreactivity was associated with the membranes of enlarged vacuoles at both 14 and 21 days (Fig. 6, E and F) in addition to staining of meiotic cells at 14 days. In control testes, TfR immunoreactivity was also seen on spermatocytes, interstitial cells, and on the basement membrane of the seminiferous tubules at both 14 days (Fig. 6G) and 21 days (data not shown), as reported previously [24, 26]. In mutant testes, TfR antibodies stained the basement membrane and some spermatocytes at both ages in the mutant testes, and they also stained the vacuolar membrane at both ages (Fig. 6, H and I). Aggregated material inside vacuoles showed modest Tf and TfR staining at 14 days (Fig. 6, E and H).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We demonstrated that the vacuoles are located in the cytosol of Sertoli cells and may represent abnormally swollen endosomes based on their positive staining for Tf and TfR, which are proteins associated with endosomal structures. These endosomal structures may be derived from the plasma membrane, because they contain membrane proteins clusterin and N-cadherin, ß-catenin (a cytosolic binding protein for N-cadherin), and actin. By electron-microscopic investigation, we also have identified what look like morphologically intact adherens junctions on the vacuole membrane. Inpp5b function may be important for the initial establishment of recycling of adherens junctions during early stages of spermatogenesis, because the vacuoles appear exactly when the developing germ cells enter meiosis I and begin to be translocated to the lumenal compartment of the epithelium. The abnormal endosomes may be prevented from recycling back to the plasma membrane to new sites of cell-cell contacts required during migration of maturing germ cells toward the lumen of the tubules. This mechanism would be consistent with the mutant phenotype, which includes detachment of developing germ cells from the seminiferous epithelium and sloughing into the epididymis. Of note, N-cadherin immunostaining was still detectable around the few vacuoles seen in the 3-mo-old mutant testis that had recovered from the initial crisis, but overall, staining with N-cadherin at this age appeared to be normal (data not shown). The recovery of spermatogenesis between 2 and 5 mo in the mutant testes, accompanied by the nearly complete recovery of a normal N-cadherin immunostaining pattern, may represent the effect of compensation by another factor, such as another 5-phosphatase.
We have gone on to study various components of endocytosis in the mutant testes by examining the localization of Sertoli cell proteins whose targeting is established (i.e., TfR, Tf, and clusterin). In carrying out these studies, it must be kept in mind that the polarity of the seminiferous epithelium is reversed from that of other epithelia. For example, fluid flows into, not from, the lumen of the tubule. Apical distribution of some proteins in Sertoli cells, such as the cadherins, differs from their concentration in basolateral membranes in most epithelial cells [4]. Our results using the TfR as a marker suggest that a deficiency in Inpp5b leads to disturbances in both apical and basolateral endocytosis.
Inpp5b is a ubiquitously expressed inositol polyphosphate 5-phosphatase that hydrolyzes lipid substrates PtdIns(4,5)P2 and PtdIns(3,4,5)P3 and soluble inositol polyphosphates Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in vitro [12, 13]. It contains the 5-phosphatase catalytic domain that is conserved among the 5-phosphatase family of proteins, but lacks a Sac1 domain, which has recently been shown to act as a broad-specificity inositol phosphatase for PtdIns(3)P, PtdIns(4)P, and PtdIns(3,5)P2 [27]. We have previously shown by mRNA in situ that Inpp5b is expressed predominantly in Sertoli cells [14]. These data support the hypothesis that the disruption in spermatogenesis in Inpp5b-/- animals is the result of a primary defect in Sertoli cells and not from an initial effect on germ cells that would then result in vacuolization of Sertoli cells. By regulating levels of 5-phosphorylated phosphoinositides, Inpp5b could play a role at multiple steps in endocytosis, including regulation of small GTPases or the actin cytoskeleton. One of the small GTPases, ARF6, which regulates coat formation and disassembly, has specifically been implicated in the internalization and recycling of plasma membrane proteins [28]. Interestingly, expression of dominant active ARF6 in HeLa cells results in a remarkably similar phenotype to that in Inpp5b-/- animals, with accumulation of actin-coated, cadherin- and ß-catenin-positive vacuoles in the cell that also contains high levels of PtdIns(4,5)P2 [29]. Phosphoinositides can act either upstream or downstream of ARF6 by recruiting and regulating ARF guanine nucleotide exchange factors or GTPase-activating proteins (GAPs) [30]; alternatively, ARF proteins can activate phosphatidylinositol 4-phosphate 5-kinase [31]. Therefore, disturbed ARF functioning and slow turnover of PtdIns(4,5)P2 could result in the inability to remove actin coating the endosomes and, in turn, produce downstream effects. This would be analogous to the phenotype seen in mice deficient in synaptojanin-1 [8].
Inpp5b could also exert its function in endocytosis through regulation of the actin cytoskeleton [11, 32]. The adhesive function of cadherin-catenin complexes and their disruption requires association of these complexes with the actin cytoskeleton and modulation of the cytoskeleton. In epithelial cells, constitutive loss of the apical cortical actin ring results in internalization of cadherin and catenin complexes [33]. PtdIns(4,5)P2 has been shown to control membrane functions by increasing and decreasing the adhesion between actin-based cortical cytoskeleton and plasma membrane, presumably at the sites of complexes between cadherin and catenin [34]. Actin-bundling proteins 
-actinin and vinculin, which are localized to adherens junctions, are known to bind PtdIns(4,5)P2. Thus, elevated PtdIns(4,5)P2 levels in Inpp5b-deficient mice could result in local disturbances in the regulation of actin polymerization at adherens junctions, resulting in internalization of cadherin-catenin complexes.
Although it is reasonable to hypothesize that the phenotype in the mutant animals results from elevated levels of 5-phosphorylated phosphoinositides in the Sertoli cell membranes of Inpp5b-deficient animals, another function of Inpp5b could be involved in the phenotype. In fact, we found wide variability in phosphoinositide levels in cultured Sertoli cells, and we were not able to demonstrate consistent elevations in cells derived from mutant animals (unpublished data). Inpp5b contains a domain in the C-terminus of the protein that has high homology to Rho-GAP domains in other proteins. Cadherin localization to cell junctions also requires the actin-reorganization activities of members of the Rho family of small GTPases, because blocking Rho and Rac in keratinocytes results in internalization of cadherin from cell-cell contacts and depolymerization of actin [35]. Rho and Rac have also been shown to interfere with endocytosis in other cell types [36]. Furthermore, Akhtar and Hotchin [37] recently reported that overexpression of constitutively active Rac1 in keratinocytes resulted in accumulation of E-cadherin and ß-catenin in large intracellular vesicles that they characterized as recycling endosomes. Therefore, loss of Inpp5b function may possibly affect other aspects of endosome cycling beyond its role as a PtdIns(4,5)P2 5-phosphatase and, thus, may have a dual function as a regulator of both PtdIns(4,5)P2 levels and the activity of certain GTPases, such as the Rho family, in the cell.
Infertility is a pleiotropic manifestation of Inpp5b deficiency. At the onset of spermatogenesis, cellular loss in the mutant tubules is due to sloughing of immature spermatocytes and spermatids rather than to excessive apoptosis. We suggest that this defect is, in turn, the result of a defect in adherens junctions and is manifested by the accumulation of swollen endocytic structures due to a block in junction recycling. Later, between 2 and 5 mo, an apparent histological recovery of the seminiferous tubules is observed in the Inpp5b-/- mice, but without the return of fertility. In these older mice, germ cell loss occurs due to excessive apoptosis as well as to defects in sperm maturation and function [14]. This complex and multifaceted phenotype resulting from a defect in a PtdIns 5-phosphatase underscores the critical role played by phosphoinositides and their metabolizing enzymes in male fertility.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Division of Intramural Research, National Human Genome Research Institute. 
2 Correspondence: Robert L. Nussbaum, Genetic Diseases Research Branch, National Human Genome Research Institute, National Institutes of Health, 49 Convent Drive, Building 49, Room 4A72, Bethesda, MD 20892-4472. FAX: 301 402 2170; rlnuss{at}nhgri.nih.gov
3 Current address: CuraGen Corporation, 322 East Main Street, Branford, CT 06405
Accepted: December 13, 2001.
Received: September 20, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2-macroglobulin, and testins: biosynthesis and differential regulation by germ cells. Mol Cell Endocrinol 1992; 89:127-140[CrossRef][Medline]This article has been cited by other articles:
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  S. Mhaouty-Kodja, A. Lozach, R. Habert, M. Tanneux, C. Guigon, S. Brailly-Tabard, J.-P. Maltier, and C. Legrand-Maltier Fertility and spermatogenesis are altered in {alpha}1b-adrenergic receptor knockout male mice J. Endocrinol., November 1, 2007; 195(2): 281 - 292. [Abstract] [Full Text] [PDF]
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 L. M. Ooms, C. G. Fedele, M. V. Astle, I. Ivetac, V. Cheung, R. B. Pearson, M. J. Layton, A. Forrai, H. H. Nandurkar, and C. A. Mitchell The Inositol Polyphosphate 5-Phosphatase, PIPP, Is a Novel Regulator of Phosphoinositide 3-Kinase-dependent Neurite Elongation Mol. Biol. Cell, February 1, 2006; 17(2): 607 - 622. [Abstract] [Full Text] [PDF]
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 J. C. Schimenti, J. L. Reynolds, and A. Planchart Mutations in Serac1 or Synj2 cause proximal t haplotype-mediated male mouse sterility but not transmission ratio distortion PNAS, March 1, 2005; 102(9): 3342 - 3347. [Abstract] [Full Text] [PDF]
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