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Biology of Reproduction 64, 13-20 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Effects of Acute Graded Reductions in Testicular Blood Flow on Testicular Morphology in the Adult Rat1

Anders Bergh2,b, Ola Collinc, and Erik Lissbrantb

b Departments of Pathology and c Anatomy, Umeå University, S-90187 Umeå, Sweden

ABSTRACT

The effect of moderate reductions in testicular blood flow has not been studied systematically. The aim of this study was, therefore, to examine the effects of different degrees of blood flow reduction on testicular morphology and to determine how much flow can be reduced before damage occurs. The subcapsular testicular artery was partially ligated in the left testes of adult rats. Testicular blood flow was measured before, immediately after, and 5 h after the ligation using laser Doppler flowmetry. After 5 h of partial ligation, the testes were removed, and their morphology was examined and related to the degree of blood flow reduction. The number of in situ end-labeled- or TUNEL-positive (i.e., dying) germ cells and the volume density of intravascular polymorphonuclear (PMN) leukocytes were measured. When flow was reduced to approximately 70% or less of its pretreatment value, a dose-related increase in the number of dying spermatogonia and early spermatocytes was seen. The PMN leukocytes accumulated in testicular blood vessels after partial ligation, and the maximum number was observed in testes where flow was reduced by approximately 50% of the pretreatment value. In conclusion, early stages of spermatogenesis are sensitive to a moderate, acute reduction in blood flow. Discrete reductions in flow may, therefore, have a large impact on sperm production.

apoptosis, spermatogenesis, testis

INTRODUCTION

In the testis, millions of sperm are produced daily. Several observations suggest that this is accomplished in an organ where the safety margin for disturbances in the vasculature could be particularly narrow [1, 2]. The testis receives its blood supply by an unusually long artery [3]. The vascular resistance in this vessel is, therefore, high, leaving capillary pressure in the testis lower than that in all other organs and only marginally higher than the venous pressure [4]. The oxygen tension in the testis is low [3, 5]. The high metabolic activity in the seminiferous tubules is apparently adapted to this environment of low oxygen and low vascular perfusion pressure, and under normal conditions, the vasculature is able to supply the testis with sufficient amounts of nutrients and oxygen. This situation suggests, however, that moderate disturbances in the blood supply to the organ could cause testicular malfunction. This poses two questions: how common are conditions in which testicular blood flow is compromised, and what is the effect of a sustained, moderate reduction of testicular blood flow?

Lumen-narrowing sclerosis is common in the testicular artery of both young and elderly men [6–8]. In spontaneously hypertensive rats, lumen obliteration is seen particularly early in the disease process in the testicular artery [9]. The testis has a limited capacity to autoregulate its blood flow, and reductions in systemic blood pressure are, therefore, accompanied by reductions in testicular blood flow [10, 11]. In line with this, circulatory shock is followed by damage to the spermatogenetic epithelium [12], and treatment of dogs with blood pressure-reducing drugs may induce seminiferous tubule damage [13]. Potent vasoconstrictors such as endothelin-1 and serotonin are produced locally in the testis, and it has been suggested that pathological overproduction of these substances may be involved in testicular malfunction [14, 15].

Several investigators have examined the morphological effects of short-term total ligation of the testicular artery followed by reperfusion. Total ligation for up to 60 min results in no or limited damage to the spermatogenetic epithelium [16–18], but ligation for 90–105 min causes selective damage to spermatogonia and preleptotene spermatocytes [16, 17]. Recent studies have shown that this cell loss is caused by apoptosis [19], and that it largely is the result of free-radical damage during the reperfusion phase [20]. A direct effect of hypoxia, however, may also be involved [21]. The morphological appearance of the spermatogenetic epithelium in infertile or elderly men is reported to be similar to that observed after experimental ischemia [7, 22, 23].

Very few experimental studies have examined the effects of moderate, sustained reductions in blood flow. Markey et al. [22, 24], however, induced partial lumen-occluding arteriosclerosis in the testicular artery of rams. They observed focal damage to the seminiferous tubules and an interstitial inflammatory reaction similar to that seen in the testes of infertile men. These studies suggest that reductions in blood flow could play an important role in the pathogenesis of male infertility. Markey et al. did not, however, measure blood flows, so it is still unknown whether such degenerative changes are the result of major or minor reductions in flow. The aim of this study was, therefore, to induce graded decreases in testicular blood flow and to examine the threshold for testicular damage.

MATERIALS AND METHODS

Animals

Adult male Sprague-Dawley rats (300–400 g) purchased from Möllegaard, Denmark, were held in standard laboratory conditions with food and water provided ad libitum. Anesthesia of the rats with pentobarbital (50–60 mg/kg i.p., Mebumal Vet, Nordvacc, Sweden) preceded the experiments. To maintain a stable body temperature (37°C) during procedures, the animals were kept supine on a heating pad.

Ligation

The scrotal sacs were opened, and the testes were exposed. A few drops of local anesthetic (Xylocain, 20 mg/ml; Astra, Södertälje, Sweden) were placed over the left subcapsular testicular artery, approximately 1 cm below its entrance into the testis. Ligation was performed by perforating the capsule immediately next to the artery with a suture needle and tying a silk 6-0 suture around the artery and a steel rod with a diameter of 290–300 µm (~70% of the diameter of the artery). The steel rod was thereafter pulled out, and the surface of the testis was gently rinsed with a few drops of saline. Testicular microvascular blood flow was measured in four different sites (near the cranial and caudal pole on the ventral and lateral aspect of the testis, respectively) before and immediately after ligation. The scrotal sacs were then closed with silk sutures, and the animals were allowed to awaken. Five hours after the ligation, the animals were again sedated with pentobarbital, and blood flow was measured in the ligated testis as described above.

Measurements of Microvascular Blood Flow

Testicular microvascular blood flow was measured using a two-channel laser Doppler flowmeter, PF 4001 master (Perimed AB, Stockholm, Sweden), and multireceiver probes, PF 412 (Perimed AB), as previously described [10]. The probes, which measure flow in a tissue volume of approximately 2 mm3, were held with a micromanipulator approximately 1 mm above an area of the testicular surface devoid of large vessels. The laser Doppler flow signals were recorded and later analyzed by a personal computer using the software Perisoft for Windows 1.01 (Perimed AB). At each time point, a mean was calculated from the four measuring sites in each testis. Averaging the laser Doppler values from multiple sites in a tissue has previously been shown to give a reasonable estimate of blood flow to an organ [25]. The laser Doppler flow signal (in arbitrary perfusion units, PFU) was also measured in the testes immediately after they were removed from the animal. In such testes, the signal is close to, but is not, zero because of persisting activity of the microvasculature [26]. In our experiments, this "biological zero" was 15 ± 2 PFU (n = 7), and it was subtracted from the measured flow values in perfused testes.

Testis Morphology

After measuring blood flow, both testes were removed. Parts of the testes were fixed in buffered formalin and embedded in paraffin, whereas other parts were fixed in 4% (v/v) formaldehyde, 3% (v/v) glutaraldehyde, and 0.05% (w/v) picric acid in cacodylated buffer and embedded in Epon as previously described [27]. Testicular morphology was examined by light microscopy of 4-µm-thick hematoxylin and eosin-stained paraffin sections or on 1-µm-thick toluidine blue-stained Epon sections. The semithin plastic embedded sections were used for studying details in the seminiferous epithelium, particularly the appearance of degenerating germ cells [28, 29].

Four-micrometer-thick, paraffin-embedded, whole-testis cross-sections were cut, and two related methods were used to stain cells with fragmented DNA (i.e., apoptotic or necrotic cells). Using two different enzymes, DNA-polymerase 1 or terminal deoxynucleotidyl transferase (TdT), labeled nucleotides were incorporated into DNA breaks and then detected by immunohistochemistry. The in situ end-labeling method (ISEL; using DNA polymerase 1) [30] was performed principally as described previously [31]. In the present study, however, the sections were digested for only 4 min, and as a detection system, 3-amino-9-ethyl-carbazole was used. The TUNEL method [32] was performed with a kit (In Situ Cell Death Detection Kit, POD; Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol. Briefly, after proteinase K treatment for 10 min, the sections were incubated at 37°C with TdT for 60 min. The number of ISEL- or TUNEL-positive germ cells (i.e., the number of dying germ cells per tubule cross-section) was then counted (100 randomly chosen, round cross-sections per testis were examined).

The volume density of polymorphonuclear (PMN) leukocytes in randomly selected testicular blood vessels was measured with the aid of a square lattice in the eyepiece of the microscope using stereological methods as previously described [33]. In summary, the number of grid intersections falling over intravascular PMN leukocytes and vascular lumina was counted in numerous random visual fields in each testis. The ratio between total hits over PMN leukocytes and total hits over vascular lumina gives an estimate for the percentage of vascular volume that is occupied by PMN leukocytes.

Statistics

Values are expressed as the mean ± SD. Groups were compared using the Mann-Whitney U-test or Student's t-test, and correlations were calculated using the Spearman rank correlation coefficients (rs). A P value of less than 0.05 was considered to be significant. In scatter plots where the number of TUNEL-positive germ cells or the intravascular PMN leukocyte concentration was plotted against blood flow, the best curve fit was calculated using the weighted least square method. For this purpose, the software STATISTICA 6.0 (Stat-soft, Tulsa, OK) was used.

RESULTS

Microvascular Blood Flow

Before ligation, average testicular blood flow was 225 ± 40 PFU (n = 37). Probably depending on how tight the knot was tied, ligation of the testicular artery resulted in a blood flow reduction that varied between animals. In some animals, flow was almost zero directly after ligation, whereas in others, it was more or less unaffected. In some (n = 28), flow was more than 10 PFU lower immediately after ligation than at 5 h after ligation, whereas in others (n = 9), flow was similar at the two time points. This suggests that the lumen of the artery in some testes was narrowed not only by the ligature but also by posttraumatic vascular spasm. Average flow directly after ligation was 0.54 ± 0.40 (n = 37) of that 5 h later, and the two flow values were correlated (rs = 0.79).

After 5 h, flow was dramatically reduced in some testes, whereas in others, no long-term effect was seen. In a sufficient number of animals, flow was moderately decreased, making it possible to relate testicular morphology to the 5-h blood flow value (see below). Vasomotion (i.e., rhythmical variations in local microcirculatory flow) was observed before ligation in all animals, but it was often absent immediately after surgery. After 5 h, vasomotion was still absent in all testes with a flow of less than 130 PFU (n = 14), but it was normal in all testes with a flow of greater than 200 PFU (n = 12). For testes with flow values of between 130 and 200 PFU, 6 of 10 testes had normal vasomotion.

General Testicular Morphology

Testicular weight was not influenced by a reduction in testicular blood for 5 h, not even when the reduction was major (data not shown). In testes with a large reduction in flow at 5 h (flow < 50 PFU), testicular morphology was, however, clearly affected, and numerous degenerating tubules (Fig. 1) and Leydig cells were observed. In testes with a more moderate reduction in flow at 5 h (flow 50–150 PFU), occasional degenerating germ cells were seen, but Leydig cells appeared to be largely unaffected. An increased number of leukocytes was observed in testicular blood vessels, but only a few had migrated into the interstitial space (Fig. 2; see below). In testes with normal or only marginally reduced flow (flow > 150 PFU), testicular morphology appeared to be unaffected.



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  		FIG. 1. Sections from testes where the artery was ligated for 5 h (a) and from the contralateral control testis (b). In a, blood flow was markedly reduced (15 PFU, 7% of pretreatment) at 5 h. Numerous degenerating tubules are observed in a but not in b. Bar = 200 µm.



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  		FIG. 2. Toluidine blue-stained Epon section demonstrating degenerating germ cells (a, arrows) in the periphery of a seminiferous tubule in a testis where the artery was partially ligated and the 5-h flow value was 95 PFU (53% of pretreatment). Occasional degenerating germ cells could also be observed in the contralateral, nonligated testis, although their number was very low (b, arrows). Leukocytes are observed in the blood vessels in a (arrowheads) but not in b. Bar = 25 µm.

Number of Dying Germ Cells in Relation to Microvascular Blood Flow

Results of TUNEL or ISEL staining of cells with fragmented DNA showed that reductions in testicular blood flow induced germ cell death. The TUNEL and ISEL staining patterns were very similar, but the TUNEL method stained slightly more cells than ISEL. The two labeling indices were highly correlated (rs = 0.87, n = 33). The stained germ cells were observed among spermatogonia and primary spermatocytes in the periphery of the tubules. In testes with a major reduction in flow (<50 PFU) at 5 h, the number of TUNEL-positive germ cells per tubule cross-section was up to 100-fold higher than that in testes with a normal flow. In several tubules, almost all germ cells in the periphery of the tubule were stained. In testes with flows of less than 50 PFU, some TUNEL-positive Leydig cells were also observed. In testes with a more moderate reduction in flow (50–150 PFU) at 5 h, only occasional germ cells at the periphery of some tubules were stained (Fig. 3). To examine the nature of such cells, the morphology of the seminiferous tubules was studied in detail in semithin, toluidine blue-stained Epon sections. In these sections, we observed single spermatogonia and primary spermatocytes with condensed, dark-staining nuclei but with normal-appearing cytoplasm (Fig. 2), that is, with a morphology similar to that described for apoptotic germ cells [29].



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  		FIG. 3. Sections stained with the TUNEL method to demonstrate dying cells (cells with fragmented DNA). a) Several labeled germ cells are observed (arrows) in a testis where blood flow was markedly reduced (70 PFU, 42% of pretreatment) after 5-h ligation of the testicular artery. b) Fewer labeled germ cells are seen (arrows) in a testis where the decrease in flow was more moderate (133 PFU, 73% of pretreatment). c) In the contralateral testis, only few dying cells are observed (arrow). Bar = 40 µm

When the number of TUNEL-positive (or ISEL; data not shown) germ cells was plotted against the measured blood flow value (PFU) or the relative reduction (5-h posttreatment value divided by the pretreatment value), it became apparent that a blood flow of lower than approximately 150 PFU (~70% of the pretreatment value) resulted in a dose-related increase in germ cell death (Figs. 3 and 4). The 5-h flow value was correlated to the number of TUNEL-positive germ cells (rs = -0.80, n = 37). For testes with flows ranging between 60% and 80% of that before ligation, germ cell death was, on average, 1.8-fold higher than that in those testes where flow was unaffected (>=100% of pretreatment value) by ligation (P < 0.05; Fig. 4). The number of TUNEL-positive germ cells was larger in the ligated than in the contralateral control testis in 18 of 19 testes with absent vasomotion. In control testes, most tubules did not contain TUNEL-positive germ cells. A few tubule cross-sections, however, contained one to three labeled germ cells, but never more than three (as previously reported [19]).



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  		FIG. 4. a) Scatterplot showing the number of TUNEL- positive germ cells per tubule cross-section (10 log) and testicular blood flow value at 5 h (PFU) in individual testes where the artery was partially ligated for 5 h. b) Scatterplot showing the number of TUNEL-positive germ cells per tubule cross-section (10 log) and relative testicular blood flow (5-h value/pretreatment value) in individual testes where the artery was partially ligated for 5 h. Pretreatment blood flow was 225 ± 40 PFU (n = 37). The number of TUNEL-positive germ cells in the contralateral unligated testes was 0.23 ± 0.08 (n = 37). Testes with or without normal vasomotion are indicated by open and filled circles, respectively.

In testes where flow was more reduced immediately after than at 5 h after ligation (n = 28), we do not know exactly at what time point flow approached the 5-h value. To elucidate this further, we examined the 18 testes in which flow immediately after ligation was less than 50 PFU. Five of these testes showed no increase in the number of TUNEL-positive germ cells, and these five testes all had a 5-h flow of greater than 150 PFU. This suggests that a transient decrease in flow does not induce germ cell death. The average values of the flow at 0 and 5 h were also calculated and plotted against the number of TUNEL-positive germ cells. In line with the results obtained when only the 5-h flow value was used, a dose-related increase clearly occurred in the number of TUNEL germ cells when the average flow was lower than approximately 130 PFU. Also, the average flow was correlated to the number of TUNEL-positive germ cells (rs = -0.77, n = 37).

Number of Intravascular Leukocytes in Relation to Microvascular Blood Flow

The highest number of intravascular leukocytes was observed for flow values of approximately 100 PFU at 5 h (Fig. 5). For flows of less than or more than this value, the number of leukocytes was lower, and the lowest values were found for flows of less than 30 and greater than 150 PFU. For the testes with 5-h flows in the range of 80% to 100% of the pretreatment value, the intravascular PMN leukocyte concentration was 2.3-fold higher (P < 0.05) than that in ligated testes with normal 5-h flow (>=100% of pretreatment). When ligated testes with a normal blood flow (>=100% of pretreatment) were compared with the contralateral testes in the same animals, an increased number of intravascular leukocytes was observed (1.9 ± 0.4 vs. 0.6 ± 0.1, respectively; n = 8; P < 0.05). The leukocytes in the manipulated testes with normal flow were, however, only observed in subcapsular venules. Surgical manipulation of the testis apparently results in a slight inflammatory response in subcapsular blood vessels, but when flow is reduced, an additional inflammatory response in all parts of the testis occurs.



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  		FIG. 5. a) Scatterplot showing the volume density of PMN leukocytes in testicular blood vessels (%) and testicular blood flow value at 5 h (PFU) in individual testes where the artery was partially ligated for 5 h. b) Scatterplot showing the volume density of PMN leukocytes in testicular blood vessels (%) and relative testicular blood flow (5-h value/pretreatment value) in individual testes where the artery was partially ligated for 5. The volume density of PMN leukocytes (%) in unligated testes was 0.6 ± 0.2 (n = 37) and 1.9 ± 0.4 (n = 8) in ligated testes with normal flow. Testes with or without normal vasomotion are indicated by open and filled circles, respectively.

DISCUSSION

Capillary pressure and oxygen tension in the testis are remarkably low (see Introduction). Spermatogenesis is, consequently, adapted to a semihypoxic environment, and one advantage of this could be to avoid oxygen-radical damage to sperm DNA [3, 34, 35]. However, all actively proliferating tissues require oxygen, and it is impossible to perfuse a tissue with blood and to form interstitial fluid without a sufficient driving vascular pressure. This study was, therefore, performed to test the hypothesis that a minor, short-term reduction in testicular blood flow will result in testicular damage. Our results suggest that this is the case. A 5-h reduction in flow to approximately 70% of the normal value may be sufficient to induce death among spermatogonia and early primary spermatocytes, but because of interanimal variation, a larger set of animals is needed to define the threshold for damage more exactly. A moderate reduction in flow also results in an inflammatory response in intratesticular blood vessels, and this response may occur at an even lower threshold. In line with previous studies, a major reduction in flow for 5 h causes substantial necrotic damage to the testis [18, 36].

For a long time, it has been recognized that total ischemia results in tissue necrosis. More recent studies show that low-grade ischemia may also induce apoptosis. For example, after compromise of blood flow to the heart or brain, both necrotic and apoptotic cell death occurs [37, 38]. The testis may respond to insufficient blood supply in a similar way. It is well known that high-grade ischemia causes necrotic damage to all parts of the testis (see above). The present study suggests that low-grade ischemia induces death among single spermatogonia and spermatocytes, and from the morphological appearance of these cells (i.e., TUNEL- or ISEL-positive single cells looking apoptotic by morphological criteria), we suggest that they die by apoptosis rather than by necrosis. The finding that these cell types may be particularly susceptible is in line with previous observations suggesting that they are the ones most sensitive to short-term total ischemia followed by reperfusion (see Introduction). In this study, we did not examine the long-term effects of a minor reduction in flow. However, because each spermatogonium (or primary spermatocyte) gives rise to a large number of sperm, it is not unlikely that a discrete reduction in spermatogonial numbers will, in time, result in a pronounced reduction in sperm production. In line with this, a threefold increase in the number of apoptotic spermatogonia and primary spermatocytes induced by 1 h of testicular torsion results, within 30 days, in an approximately 50% drop in daily sperm production [19]. In the present study, the number of dying germ cells was increased 1.8-fold in testes with flows of 60%–80% of the pretreatment value, suggesting that a discrete but sustained reduction in flow could have a major impact on sperm production. Further studies are needed to test this hypothesis. Our own unpublished data, however, show a 50% decrease in testicular weight after 2 wk in testes where flow is reduced to approximately 65% of the pretreatment value.

A reduction in testicular blood flow was associated with an increase in the number of leukocytes in testicular blood vessels, and occasional leukocytes migrated into the interstitial space (i.e., an early inflammation-like response was induced). This response was not seen in testes where flow was reduced to, or close to, zero, probably because this prevents the influx of leukocytes. Tissue necrosis damages cellular membranes, and the released intracellular material induces an acute inflammatory response. This leukocyte accumulation is necessary for subsequent tissue healing. Accumulating leukocytes may also cause additional oxygen radical-mediated damage to the tissue, particularly if flow returns to a damaged tissue [39]. In this study, it is, therefore, not surprising that we observed leukocyte accumulation in testes where we also noted signs of tissue necrosis, that is, in testes with a major decrease in flow. In testes where flow was approximately 50%–75% of the pretreatment value, however, no apparent signs of necrosis were observed. In such testes, single germ cells were dying, apparently by apoptosis, and one of the principal characteristics of apoptosis is that it does not induce inflammation. Moreover, intravascular leukocyte accumulation was also observed in testes where flow had been only slightly reduced as well as in testes without increased germ cell death. This may suggest that leukocyte accumulation in the testes is not always secondary to germ cell death. During reperfusion after short-term (i.e., 1-h) testicular torsion, leukocytes accumulate in the testis, possibly as a result of ischemia-induced up-regulation of endothelial adhesion molecules [19]. As radical scavengers reduce testicular damage after torsion followed by reperfusion, it is likely that free radicals from leukocytes can contribute to germ cell apoptosis [19, 20]. It is, therefore, possible that a sustained, moderate reduction of testicular blood flow may cause germ cell death by two different mechanisms: effects caused by hypoxia directly, and effects caused by accumulating leukocytes. Their relative importance and the pathogenic mechanisms by which hypoxia and oxygen radicals damage testicular cells, however, remain to be elucidated. The reason why a discrete reduction in testicular blood flow apparently up-regulates endothelial cell adhesion molecules, causing intravascular leukocyte accumulation, is also unknown. Previous studies have, however, shown that the testis constitutively secretes inflammation mediators. Testicular interstitial fluid causes an acute inflammation when injected subcutaneously, and the effects of these mediators are, under normal conditions, balanced by the local secretion of inflammation inhibitors [40]. Is this inhibitor down-regulated by low-grade ischemia?

In testes where the blood flow reduction was sufficient to induce germ cell death and an inflammation-like vascular response, normal vasomotion was generally not seen. The reason for this is unknown, but at least two factors may be involved. Vasomotion could depend on a sufficient flow and intravascular pressure. However, the disappearance of vasomotion may also be a sensitive marker of testicular malfunction, both because previous studies have demonstrated disturbed vasomotion in testes with tubule damage induced by cryptorchidism or irradiation [41] and because normal vasomotion does not reappear after repair of 1-h testicular torsion, even when flow is normalized [42]. In the testis, vasomotion is important for transvascular fluid exchange [2], and hypothetically, inhibition of vasomotion could, therefore, contribute to the testicular malfunction.

ACKNOWLEDGMENTS

The authors wish to thank Mrs. Sigrid Kilter, Birgitta Ekblom, and Elisabeth Dahlberg for their fine technical assistance.

FOOTNOTES

First decision: 8 June 1999.

1 Supported by grants from the Swedish Medical Research Council (project 5935), the Maud and Birger Gustavsson foundation, Swedish Match, and the Medical Faculty at Umeå University. Back

2 Correspondence. FAX: 46 90 7852829; anders.bergh{at}pathol.umu.se Back

Accepted: July 20, 2000.

Received: May 12, 1999.

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