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a C.I.R., Facultad de Medicina, Universidad de Buenos Aires, 1121 Buenos Aires (UBA), Argentina
b Department of Pathology and Cell Biology, Faculty of Medicine, Université de Montréal, Montréal, P. Québec, Canada H3T 1J4
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dym et al. [12] reported that when Sertoli cells were cultured on basement membrane substrates, Gs complexes of adenyl cyclase and the cAMP response to FSH were enhanced, a response that is consistent with morphologically and functionally differentiated Sertoli cells. However, the same authors also reported that when soluble laminin was added to Sertoli cells grown on plastic, no change in the intracellular cAMP accumulation was observed, suggesting that besides the interaction of extracellular matrix components, a number of other interrelated factors could be responsible for inducing changes in the Sertoli cell's morphology and function. The laminins constitute the major noncollagenous basement membrane component. These basement membrane proteins have been the focus of numerous studies (reviewed in [13]). In organ culture, antibody inhibition of laminin has been reported to modify basement membrane deposition and epithelial morphogenesis [14–17]. There is evidence that basement membranes are required for epithelial cell differentiation and other cellular functions.
We have previously demonstrated that antibodies against a preparation enriched in basement membranes of seminiferous tubules (STBM) [18] or a noncollagenous fraction of STBM [19] passively transferred induced modifications to the basement membranes and focal sloughing of the seminiferous epithelium in the rat. In the present report, we tested the effect of passive immunization with anti-laminin IgG on the limiting membrane of the seminiferous tubules, on germ cell differentiation, and on the competence of the Sertoli cell junctions forming the blood-tissue barrier in the adult guinea pig.
The data presented here show that passive immunization with anti-laminin IgG caused a thickening of the limiting membrane of the seminiferous tubules coincident with the sloughing of spermatids and of older classes of spermatocytes, but it allowed Sertoli cell tight junctions that form the blood-testis barrier to remain competent in the guinea pig. Immunofluorescence-positive deposits of rabbit IgG were detected in the limiting membrane of seminiferous tubules and within the walls of the blood vessels located in the interstitial space of the testes of treated animals. By 80 days after the onset of passive transfer of antibodies, the seminiferous epithelium showed characteristic features of normal guinea pig. This study provides an in vivo model for investigating the role of the extracellular matrix proteins in the maintenance of spermatogenesis and in the structural integrity of the seminiferous epithelium.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mouse laminin-l (Gibco BRL, Life Technologies, Grand Island, NY) obtained from the extracellular matrix of a murine Engelbreth Holm-Swarm (EHS) sarcoma was used as antigen. Two rabbits received intradermal injections of an emulsion of complete Freund's adjuvant and laminin (0.4 mg). They received a total of three injections with 15-day intervals between each one. The IgG was isolated from the rabbit sera as previously described [20]. Anti-laminin IgG was further purified by affinity chromatography over a column of mouse laminin-1 Sepharose.
Antibodies against laminin-l with a titer of 1 x l06 were detected by an ELISA. No cross-reaction was detected by ELISA when anti-laminin IgG was reacted with type IV collagen obtained from EHS sarcoma (Gibco) or rat fibronectin (Gibco) [20]. IgG obtained from normal rabbit serum (NRS) served as control.
Passive Transfer of Antibodies
A total of fifty-one 340- to 440-g adult male guinea pigs were used in this study. They were divided into three groups of 17 animals: group 1) control nonimmunized; group 2) control, injected i.v. with 25 mg of normal rabbit IgG; and group 3) experimental, injected i.v. with 25 mg of rabbit anti-laminin IgG. Guinea pigs were killed at 12, 20, 30, 50, and 80 days after the first injection. Guinea pigs killed at 30, 50, and 80 days received the same immunization schedule, i.e., three injections: one on Day 0, one on Day 10, and one on Day 20. Animals killed at 12 days received one injection at Day 0, and those killed at 20 days received two injections, one at Day 0 and another at Day 10. Testes were removed and processed for light and electron microscopy and immunohistochemistry.
Light Microscopy of Paraffin Sections
The testes were fixed by perfusion of 15 ml of PBS pH 7.4, followed by 60 ml of Bouin's fixative through the testicular artery. Perfusion-fixed testes were further immersed-fixed in the same fixative mixture for an additional 36–48 h at room temperature (RT) [21, 22] and dehydrated in ethanol before being cleared in xylene for paraffinization.
Electron Microscopy of Thin Sections
The testes were perfused through the testicular artery [23] with PBS (154 mM NaCL, 100 mM sodium phosphate pH 7.3) followed by 60 ml of a solution of 5% glutaraldehyde buffered with 0.1 M sodium cacodylate at pH 7.3 and containing 0.05% CaCl2. The fixed tissues were diced into 1-mm3 pieces and further immersed for 6–8 h in the same fixative. The tissue pieces were washed in the same buffer, postfixed for 6–8 h at RT in a solution of 1% OsO4 in Veronal acetate [24], and stained en bloc in a solution of 5% uranyl acetate in Veronal acetate buffer pH 4.5–5.2. The remaining tissue were processed through the potassium ferrocyanide-tannic acid-uranyl acetate staining en bloc or PFTA technique as described earlier [25]. Dehydration was carried out in a graded series of ethanol. Tissue pieces were embedded in PolyBed 812 (PolySciences Ltd., Warrington, PA). Semithick sections (0.5 µm) stained with a solution of 1% toluidine blue or Azur II were used for observation with a Leitz Ortholux II light microscope (Leitz, New York, NY). Sliver-thin sections were cut with a diamond knife, collected on formvar-coated carbon stabilized grids, and examined at 80 kv with a Zeiss EM 9A (Carl Zeiss, Inc., Thornwood, NY) or a Philips 300 electron microscope (Philips, Eindhoven, The Netherlands).
Quantification Studies
The variations in the diameters of seminiferous tubules and of their lumina after passive immunization with anti-laminin IgG were recorded on 0.5-µm semithick toluidine blue-stained sections using an ocular micrometer and a Leitz Ortholux II light microscope. The diameters of the tubules and of their lumina were found to vary little during spermatogenesis from one stage of the cycle of the seminiferous epithelium to the next in the normal guinea pig; therefore, the data recorded for the treated and control animals did not take into consideration the stage of the cycle. The data in Table 1 were obtained by counting the number of damaged tubules using 4–6 testicular tissue sections per block, 5 blocks per guinea pig, and a total of four testes per group for each day of treatment used. The morphometric analyses of the variations in the size of individual components within the limiting membrane (Table 2) of a total of 150 (30 per animal group) seminiferous tubules and of their ratios to one another were done on sliver-thin sections with a Zeiss EM 9A electron microscope equipped with a Zeiss modular system for quantitative digital analysis. The data shown in Tables 1 and 2 are the mean ± SEM. Data were evaluated by one-way ANOVA, and differences between means were analyzed by the Neuman-Keuls multiple-range test or Student's t-test according to the number of groups [26].
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Junctional Permeability Tracer Studies
In the present study, testes were first excised from the anesthetized animal and later infused with a solution of 30 mg of type II horseradish peroxidase (HRP; Sigma Chemical Company, St. Louis, MO) dissolved in 4 ml of Medium 199 through the internal spermatic artery. This approach represents a modification of the technique described earlier by Pelletier [27], in which testicular permeability was studied at different times following vascular infusion of HRP through the portal vein. In the current investigation, infusion of the tracer was followed by a perfusion of ~60 ml of 5% glutaraldehyde in sodium cacodylate buffer (0.1 M, pH 7.3) through the testicular artery. The fixed testes were diced into 3- to 5-mm3 fragments, immersion-fixed for an additional 90 min, and washed at 4°C with buffer containing 7% of sucrose before being sectioned into 40-µm sections with a Vibratome (series 1000 Pelco 101; Technical Products International Inc., St. Louis, MO). Vibratome tissue sections were incubated at RT, with 3–3' diaminobenzidine (DAB) + 0.01% H2O2 [28, 29]. The sections were postfixed in a solution of 1% OsO4 in Veronal acetate pH 7.3 and processed for electron microscopy. Controls included either omitting H2O2 from the incubation medium or not exposing tissues to HRP.
Detection of IgG Deposits by Immunofluorescence
To detect IgG deposits, 4- to 6-µm cryostat testis sections were fixed for 7 min in cold acetone, washed in PBS, and incubated with a goat fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (1:15) or with an FITC anti-guinea pig IgG (1:/25) (Cappel Lab., Cochranville, PA) for 40 min at RT. Tissue sections were washed in PBS and mounted in buffered glycerine. Controls included a first incubation with an NRS or a nonconjugated anti-rabbit IgG. Sections were observed with a Zeiss Axiophot microscope equipped with epifluorescence.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The variations in the diameters of the tubules and of the lumina at different time intervals after passive immunization with anti-laminin IgG are presented in Table 1. In this table, the percentages of tubules affected by the treatment, i.e., those that showed a reduction in the size of the lumen, increased up to 30 days after the first injection, with a maximum number of damaged tubules occurring at around 30 days. The number of affected tubules dropped from 73% 30 days after the first injection to 16% and 13%, respectively, 50 and 80 days after the first injection. The remaining 27% of the tubules (at 30 days after the first injection) unaffected by immunization against laminin were at different stages of the seminiferous epithelium. The testes of nonimmunized guinea pigs and guinea pigs injected with NRS IgG showed normal features at all time intervals studied. Data in Tables 1 and 2 were obtained by analyzing the testes of guinea pigs killed 30 days after the first injection of NRS IgG.
Light Microscopy
In untreated animals and control animals treated with NRS IgG, the twelve stages of the cycle of the seminiferous epithelium as defined by Clermont [30] showed all the histological features already described in the normal guinea pig, including a well-defined lumen that occupied approximately one third of the total diameter of the tubule (Fig. 1a). In the experimental groups, seminiferous tubules showed severe lesions at 12 (Fig. 1b) and 30 days (Fig. 1c) after the first injection of anti-laminin IgG. The tubules showed multiple foci of sloughing of elongated and round spermatids (Fig. 1, b and c). Degeneration of the older classes of spermatocytes (Fig. 1b) and formation of giant cells near the tubular lumen was observed. Severely damaged tubules contained young classes of spermatocytes, spermatogonia, and Sertoli cells (Fig. 1c). Mononuclear cell infiltration of the seminiferous tubules was rare. However, in most instances, lesions to the seminiferous epithelium were accompanied by a thickening of the limiting membrane of the tubule (Fig. 1c). In the interstitial tissue, a discrete peritubular and perivascular mononuclear cell infiltrate was observed (Fig. 1c). Animals receiving anti-laminin IgG injections 50 or 80 days earlier showed only rare damaged tubules (Fig. 1d).
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Quantitative Studies of the Thickening of the Limiting Membrane Constituents
A quantitative evaluation of the basal lamina was performed on thin epoxy resin-embedded testis sections by electron microscopy. Table 2 shows the variations in 1) the thickness of the total limiting membrane (LIMM) (measured from the lamina interna (LI) to the lamina externa (LE) inclusively), 2) the thickness of individual LI and LE, and 3) the variations in individual ratios of LI:LIMM and LE:LIMM measured at different time intervals after the first anti-laminin IgG injection. The data in Table 2 show that the thickness of the LIMM was maximal 30 days after the first injection of anti-laminin IgG. The appearance of the most severe lesions in the seminiferous epithelium (see previous section) coincided with the occurrence of a maximal thickness of the LIMM around 30 days after the beginning of the treatment. In addition, the data reveal that the thickening of the LI contributed most to the thickening of the LIMM.
Electron Microscopy
In controls that received injections of NRS IgG, the basal lamina constituted a thin and rather straight band of amorphous material at a short distance from the base of the Sertoli cells and situated within a narrow and uniformly sized LI (Fig. 2a). Twelve days after the beginning of anti-laminin treatment, the base of the Sertoli cells showed an irregular outline (Fig. 2b). The LI showed an increased width and contained electron opaque deposits that had a distribution similar to the one of immunofluorescence-positive immune complexes in Figure 5b. By 20 days after the first anti-laminin injection, the basal lamina showed infolding that projected into an enlarged LI (Fig. 2c) Thirty days after the beginning of the treatment, type I collagen fibrils similar in amounts and disposition to those of the control animals were seen adjacent to the basal lamina (Fig. 2, d and e). In addition, bundles of collagen fibrils with a 110-nm periodicity typical of the type VI collagen [31] were present (Fig. 2, d and e). The amounts of type VI collagen in this area seemed to be greater than those observed in controls.
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Junction Permeability Tracer Studies
In animals treated with NRS IgG, the Sertoli cell tight junctions that constitute the blood-testis barrier showed the same permeability characteristics (Fig. 3) as those documented in the normal noninjected adult guinea pig [32]. In animals treated with anti-laminin IgG, monocytes were seen within the LI and sandwiched between the myoid cells basally and the basal lamina apically (Fig. 4a). Leukocytes were not found beyond Sertoli cell tight junctions or within the depth of the seminiferous epithelium. Vacuolization of Sertoli cells and enlarged intercellular spaces between two Sertoli cells were frequent. We observed no disruption in the permeability of the blood-testis barrier after injection with anti-laminin IgG, as evidenced by the presence of competent Sertoli cell tight junctions at all time intervals studied after the injection (Fig. 4b). An interesting observation was the finding of reaction product in the intercellular clefts between aberrant but not between normal spermatids and the Sertoli cell plasma membrane (Fig. 4c). The intercellular clefts surrounding normal germ cells older than the leptotene spermatocytes were consistently devoid of reaction product (Fig. 4c). In testes of guinea pigs treated 50 and 80 days earlier with anti-laminin IgG, the permeability of Sertoli cell tight junctions was similar to that documented in the normal noninjected adult guinea pig [32].
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Immunofluorescence
Immunofluorescence-positive deposits of rabbit and guinea pig IgG were not detected in guinea pigs injected with NRS at any of the time intervals studied (Fig. 5a). However, testes from animals injected 12 and 30 days earlier with anti-laminin IgG showed immunofluorescence-positive deposits of rabbit IgG within the LIMM of numerous seminiferous tubules and within the walls of blood vessels contained in the interstitial space (Fig. 5b). Deposits of rabbit IgG were not observed in testes from guinea pigs injected 50 and 80 days earlier; faint but positive deposits of guinea pig IgG were present in the LIMM of seminiferous tubules 50 days after initiation of anti-laminin treatment (not shown). In addition, strongly positive deposits were found in the glomerular and, to a lesser degree, in the tubular basement membrane of the kidney of guinea pigs passively treated with anti-laminin at each time interval studied (not shown). Guinea pig IgG was still detected in the kidney of animals injected 50 and 80 days earlier with anti-laminin IgG (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study shows that passive immunization with anti-laminin IgG induced a thickening of the LIMM of the seminiferous tubule, especially of the LI, and that this thickening was accompanied by severe yet transient modifications in the epithelium. These modifications included an increase in the deposit of extracellular fibrils with repeating cross-striations similar to those seen in type VI collagen, the appearance of infolding in the basal lamina, vacuolization of Sertoli cells, and sloughing of spermatids and spermatocytes.
In the normal developing rat testis, type VI collagen has been reported to fill the interstitial spaces between fetal cords, but in the adult, the same type of collagen was shown confined to the LIMM of the tubules and of the adventitia of the vessels [33, 34]. Furthermore, it was concluded that type VI collagen was distributed independently from type I collagen fibrils in the interstitial spaces and that it was excluded from the basal lamina of the Sertoli cells [34], suggesting that peritubular myoid cells may be, at least in part, responsible for the deposit of type VI collagen. The Sertoli cells and myoid cells have been documented to contribute to the formation of the LI [35, 36], and the present report and our previous finding [37] that passive transfer of anti-laminin IgG induced a thickening of the LI suggest that the treatment affects both cells. It is noteworthy that the occurrence of infolding in the basal lamina is a phenomenon that was reported to accompany severe modifications of the seminiferous epithelium regardless of whether these modifications were pathological in nature, as in experimental auto-immune orchitis [38] and the present report, or natural, as in the seasonal testicular regression [27, 38–40]. Taken together, the results suggest that passive immunization with anti-laminin IgG induced changes in the laminin component of the seminiferous biomatrix that accompany and perhaps cause modifications in Sertoli cells, which no longer can provide an environment adequate for the normal progression of spermatogenesis. The maximum number of damaged seminiferous tubules occurred 30 days after the beginning of the treatment. Most assaults to the testis usually cause patchy lesions, and one cannot rule out that periods slightly longer than 30 days after treatment might reveal a greater number of affected tubules; however, the fact that the remaining 27% of the tubules unaffected by immunization against laminin were at different stages of the seminiferous cycle suggests that resistance to the experimental treatment was not stage-specific. In vitro studies have shown that the environment in which Sertoli cells are grown, namely the type of collagen and laminin, affects their morphology [9, 41] and behavior. Sertoli cells in culture have been documented to secrete type I collagen in response to reconstituted basement membrane [4]. The organization of cultured Sertoli cells has been documented to be modified by increasing the thickness of the reconstituted basement membrane [4]. Individual components of the basement membrane have been shown to affect the differentiation and survival of cultured cells (reviewed in [42]). Furthermore, the extracellular matrix substrate composition has been shown to affect the basal diffusion of medium through the basement membrane and to be a factor of crucial importance for the maintenance of differentiated monolayers of Sertoli cells [4].
The laminins constitute the major noncollagenous basement membrane component (reviewed by Timpl [13]). In organ culture, antibody inhibition of laminin binding to its cellular receptor or to other basement membrane constituents has been reported to modify basement membrane deposition and epithelial morphogenesis [14–17]. Targeting the LAMC1 gene was documented to result in embryonic lethality due to the failure of endoderm differentiation caused by an absence of basement membrane [43]. Smyth et al. [43] showed that the laminin 
1 subunit was required for laminin assembly and that laminin in turn was necessary for the organization of the other basement membrane components and for extra-embryonic endoderm differentiation in vitro as well as in vivo [43]. In the present study, passive immunization with anti-laminin IgG induced deposits of rabbit IgG within the LIMM of the seminiferous tubules and in the adventitia of the vessels. The damage to the biomatrix of the seminiferous tubule, the presence of immune complexes, and the damage to the epithelium caused by immunization are all coincidental. When the biomatrix appears as normal and without immune complexes, the epithelium is intact and spermatogenesis progresses normally, suggesting that the basement membrane needs to resume its normal structural and functional integrity for the epithelium to do the same and for spermatogenesis to be reinitiated. It is noteworthy that the deposits of rabbit IgG observed during the early phase of passive anti-laminin antibody transfer, i.e., 12–30 days after the beginning of the treatment, were no longer seen during the late phase 50 and 80 days after the beginning of the treatment, when lesions to the epithelium were no longer present and progression of spermatogenesis was normal. In testicular biopsies of infertile patients, changes in the basal lamina of the seminiferous tubules and the presence of immune complexes were reported to accompany impairment of spermatogenesis [44, 45]. These results suggest the existence of a link between the composition of the tubule biomatrix, the integrity of Sertoli cells, and the progression of spermatogenesis in vivo. Furthermore, the finding of rabbit IgG deposits in the kidney of anti-laminin-treated guinea pigs and our previous reports [18, 46] confirm that passive immunization with anti-laminin IgG induces changes not only to the testis but to other organs of the body [47, 48]. This view is supported by other reports showing that epithelial morphogenesis was inhibited after disruptions of cell-extracellular matrix interactions in the developing lung, salivary glands, and kidney [14, 16, 49].
The deposits of guinea pig IgG in the same location in which rabbit IgG was found throughout the duration of anti-laminin treatment might reveal a host response to the rabbit anti-laminin IgG injected similar to that reported during glomerulonephritis induced by passive transfer of antibodies [50, 51].
As indicated above, there is an entire body of evidence pointing to a key role of the basement membrane in the differentiation of epithelia. However, additional evidence revealed that basement membrane is not the sole factor [43] involved and that cell-to-cell interactions may also influence epithelial development [52]. In our experimental model, the sloughing of spermatids and of pachytene spermatocytes, which is an unspecific manifestation of an assault to the testis, suggests the appearance of modifications to the Sertoli cell-germ cell interactions such that postmeiotic germ cells are forced to leave the seminiferous epithelium before completion of their cellular maturation in the tubule. It may be that in the presence of a modified basement membrane of the tubule, the Sertoli cell-germ cell contacts were no longer able to retain the older germinal cells within the seminiferous epithelium. It is conceivable that antibodies to laminin may modify integrin-mediated signal transduction and thereby influence the Sertoli cell cytoskeleton that is associated with either the cell matrix or with the cell-cell junctions.
The Lumen and the Competence of the Blood-Testis Barrier
In this study, the highest percentage of tubules with reduced lumina and severe destruction to the seminiferous epithelium caused by passive immunization with anti-laminin IgG was observed around 30 days after initiation of the treatment. Although they responded to different mechanisms, the anti-laminin-induced changes we reported here in the adult guinea pig testis were reminiscent of seasonal testicular changes we reported in a seasonal breeding bird, the mallard duck [53]. In the mallard duck, 1) the lumen was not closed but rather substantially reduced in diameter, becoming occupied by a lipid plug; 2) the Sertoli cell tight junctions remained competent in blocking entry of vascularly infused permeability tracer; 3) the LIMM underwent sizeable thickening, and the older classes of germ cells were sloughed off. It is noteworthy that the annual seasonal reproductive cycle in the duck [53] and passive immunization with anti-laminin IgG in a continual breeder, the guinea pig (present report), induced a reduction in the size of the tubular lumen that was not accompanied by a closure, and in both instances there was no change in the competence of the Sertoli cell junctions that form the blood-testis barrier. Conversely, in an emphatic mammalian seasonal breeder such as the mink, in which the annual reproductive cycle involves a closure of the lumen, this phenomenon was accompanied by a change in the competence of the tight junctions of the blood-testis barrier [25, 27]. Together, these results give further support to the view that the degree of competence of the blood tissue barrier within the testis is conferred by the presence or the absence of a lumen within the seminiferous tubule rather than by the phase of cellular development of the germ cells present within the epithelium [54–56].
One interesting observation in the present study was the finding of junctional permeability tracer in the intercellular cleft between aberrant spermatids and Sertoli cells, but not in the intercellular cleft between normal spermatids and Sertoli cells. Together, the Sertoli cell tight junctions ensured a competent blood-testis barrier [56], yet allowed for creation of isolated permeated pockets that contained germ cells showing signs of degeneration. Conceivably, there would exist an up-regulation of germ cell receptors that would selectively identify degenerating germ cells to the Sertoli cells so that only the degenerating cells would be enclosed in a permeable pocket, without modifying the competence of the rest of the tissue barrier. Interestingly, the expression of the cell adhesion molecules, L-selectins, involved in leukocyte homing has been demonstrated in the testis [57]. The existence in the testis of a fas-mediated pathway in which injured Sertoli cells with reduced supporting capacity would induce a negative selection process of germ cells has been proposed [58].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by CONICET (PIP 0595) and UBA (TM32) to L.L., B.D., and R.P.; and by Population Council Award B99.049/ICMC and NSERC #OGP0041653 to R.M.P. 
2 Correspondence: R.-Marc Pelletier, Université de Montréal, Faculty of Medicine, Department of Pathology and Cell Biology, Pavillon Principal, 2900 Edouard-Montpetit blvd., Montréal, PQ, Canada H3T 1J4. FAX: 514 485 7932; pellemar{at}ere.umontreal.ca
Accepted: January 4, 2000.
Received: August 17, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6 subunit is essential for epithelial polarization in developing kidney tubules. J Cell Biol 1990; 111:1265–1273. (+) and (-) isoforms in guinea pig Sertoli cells: a possible association with F-actin and G-actin. Biol Reprod 1997; 57:367–376.[Abstract]This article has been cited by other articles:
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  R.-M. Pelletier, S. R. Yoon, C. D. Akpovi, E. Silvas, and M. L. Vitale Defects in the regulatory clearance mechanisms favor the breakdown of self-tolerance during spontaneous autoimmune orchitis Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2009; 296(3): R743 - R762. [Abstract] [Full Text] [PDF]
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 M. K.Y. Siu and C. Y. Cheng Extracellular Matrix: Recent Advances on Its Role in Junction Dynamics in the Seminiferous Epithelium During Spermatogenesis Biol Reprod, August 1, 2004; 71(2): 375 - 391. [Abstract] [Full Text] [PDF]
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J. C. Rockett, F. L. Mapp, J. B. Garges, J. C. Luft, C. Mori, and D. J. Dix Effects of Hyperthermia on Spermatogenesis, Apoptosis, Gene Expression, and Fertility in Adult Male Mice Biol Reprod, July 1, 2001; 65(1): 229 - 239. [Abstract] [Full Text] [PDF]
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