Oxygen radicals and reactive oxygen species in reproduction

Free radicals and reactive oxygen species play a number of significant and diverse roles in reproductive biology. In common with other biological systems, mechanisms have evolved to minimize the damaging effects that these highly reactive molecules can have on reproductive integrity. Conversely, however, recent findings illustrate the constructive roles that oxygen radicals and reactive oxygen species play in a number of important junctures in the development of germ cells and the obligate endocrine support they receive for the successful propagation of the species. Specifically addressed in this review are some aspects of sperm development and action, the uterine environment, oocyte maturation and ovulation, and corpus luteum function and regression.     

Role of reactive oxygen species in the response of barley to necrotrophic pathogens

Summary.  The interactions between Hordeum vulgare (barley) and two fungal necrotrophs, Rhynchosporium secalis and Pyrenophora teres (causal agents of barley leaf scald and net blotch), were investigated in a detached-leaf system. An early oxidative burst specific to epidermal cells was observed in both the susceptible and resistant responses to R. secalis, and later on, a second susceptible-specific burst was observed. Time points of the first and the second burst correlated closely with pathogen contact to the plasma membrane and subsequent cell death, respectively. HO₂ ^•/O₂ ⁻ levels in resistant and susceptible responses to P. teres were limited in comparison. During later stages, HO₂ ^•/O₂ ⁻ was only detected in 2 to 3 epidermal cells immediately adjacent to phenolic browning and cell death observed during the susceptible response. However, H₂O₂ was detected in the majority of mesophyll cells adjacent to the observed lesion caused by P. teres. In contrast to observations during challenge with R. secalis, no direct contact between P. teres and the plasma membrane at sites of reactive oxygen species production was evident. Preinfiltration of leaves with antioxidants prior to challenge with either pathogen had no effect on resistance responses but did limit the growth of the pathogens and inhibit the extent of cell death during susceptible responses. These results suggest a possible role for reactive oxygen species in the induction of cell death during the challenge of a susceptible plant cell with a necrotrophic fungal leaf pathogen. Received May 2, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Department of Plant Science, Waite Campus PMB1, University of Adelaide, Glen Osmond, South Australia 5064, Australia.     

Copper-mediated oxidative burst in Nicotiana tabacum L. cv. Bright Yellow 2 cell suspension cultures

Summary.  In cell suspension cultures of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) a rapid and concentration-dependent accumulation of H₂O₂ is induced by excess concentrations of copper (up to 100 μM). This specific and early response towards copper stress was shown to be extracellular. Addition of 300 U of catalase per ml decreased the level of H₂O₂. Superoxide dismutase (5 U/ml) induced an increase in H₂O₂ production by 22.2%. This indicates that at least part of the H₂O₂ is produced by dismutation of superoxide. Pretreatment of the cell cultures with the NAD(P)H oxidase inhibitors diphenylene iodonium (2 and 10 μM) and quinacrine (1 and 5 mM) prevented the generation of H₂O₂ under copper stress for 90%. The influence of the pH on the H₂O₂ production revealed the possible involvement of cell-wall-dependent peroxidases in the generation of reactive oxygen species after copper stress. Received May 20, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Plant Physiology, Department of Biology, University of Antwerp (RUCA), Groenenborgerlaan 171, 2020 Antwerp, Belgium.     

Reactive oxygen species in spermatozoa: methods for monitoring and significance for the origins of genetic disease and infertility

Human spermatozoa generate low levels of reactive oxygen species in order to stimulate key events, such as tyrosine phosphorylation, associated with sperm capacitation. However, if the generation of these potentially pernicious oxygen metabolites becomes elevated for any reason, spermatozoa possess a limited capacity to protect themselves from oxidative stress. As a consequence, exposure of human spermatozoa to intrinsically- or extrinsically- generated reactive oxygen intermediates can result in a state of oxidative stress characterized by peroxidative damage to the sperm plasma membrane and DNA damage to the mitochondrial and nuclear genomes. Oxidative stress in the male germ line is associated with poor fertilization rates, impaired embryonic development, high levels of abortion and increased morbidity in the offspring, including childhood cancer. In this review, we consider the possible origins of oxidative damage to human spermatozoa and reflect on the important contribution such stress might make to the origins of genetic disease in our species.     

Radicaux libres et spermatozoïdes humains: physiologie et physiopathologie

The role of reactive oxygen species in the physiopathology of human sperm function has been emphasized in recent years. Their production in semen has been associated with loss of motility, decreased capacity for spermoocyte fusion and loss of fertility. In semen preparations, there are two major sources of reactive oxygen species: leucocytes and spermatozoa themselve. It has been proposed that reactive oxygen species production by human spermatozoa was dependent upon a membrane-bound NADPH oxidase or a mitochondrial diaphorase. Hydrogen peroxide produced by the dismutation of superoxide anion has been recognized as the most toxic oxidizing species for human spermatozoa. Owing to their high content of polyunsaturated fatty acids, it has been proposed that lipid peroxidation of the sperm plasma membrane is largely responsible for defective sperm function. Reactive oxygen species also affect the sperm axoneme as a result of ATP depletion, inhibit mitochondrial functions, and synthesis of DNA, RNA and proteins, produce cytoskeletal modifications and inhibit sperm-oocyte fusion. Human spermatozoa possess enzymatic defence systems such as superoxide dismutase, glutathion peroxidas/reductase and catalase to counteract the toxic effects induced by reactive oxygen species. Correlations have been reported between their effectiveness and the duration of sperm motility. If the excessive production of reactive oxygen species is detrimental for human spermatozoa, they could also participate in the physiological function of the spermatozoa when present at low concentrations. Indeed, reactive oxygen species have been shown to be involved in the activation of several enzymes. Furthermore, sperm capacitation, acrosome reaction and sperm-zona interaction would be enhanced by reactive oxygen species.     

Continuous spermatogenesis and the germ cell development strategy within the testis of the Jamaican Gray Anole, Anolis lineatopus

Testicular tissues from Anolis lineatopus were examined histologically to determine testicular structure, germ cell morphologies, and the germ cell development strategy employed during spermatogenesis. Anoles (N = 36) were collected from southern Jamaica from October 2004 to September 2005. Testes were extracted and fixed in Trump's fixative, dehydrated, embedded in Spurr's plastic, sectioned, and stained with basic fuchsin/toluidine blue. The testes of Jamaican Anoles were composed of seminiferous tubules lined with seminiferous epithelia, similar to birds and mammals, and were spermatogenically active during every month of the year. However, spermatogenic activity fluctuated based on morphometric data for February, May and June, and September-December. Sequential increases for these months and decreases in between months in tubular diameters and epithelial heights were due to fluctuations in number of elongating spermatids and spermiation events. Cellular associations were not observed during spermatogenesis in A. lineatopus, and three or more spermatids coincided with mitotic and meiotic cells within the seminiferous epithelium. Although the germ cell generations were layered within the seminiferous epithelium, similar to birds and mammals, the actual temporal development of germ cells and bursts of sperm release more closely resembled that reported recently for other reptilian taxa. All of these reptiles were temperate species that showed considerable seasonality in terms of testis morphology and spermatogenesis. The Jamaican Gray Anole has continuous spermatogenesis yet maintains this temporal germ cell development pattern. Thus, a lack of seasonal spermatogenesis in this anole seems to have no influence on the germ cell development strategy employed during sperm development.     

New assays for detection and localization of endogenous lipid peroxidation products in living boar sperm after BTS dilution or after freeze-thawing

Reactive oxygen species have been implicated in sperm aberrations causing multiple pathologies including sub- and infertility. Freeze/thawing of sperm samples is routinely performed in the cattle breeding industries for semen storage prior to artificial insemination but unusual in porcine breeding industries as semen dilution and storage at 17 degrees C is sufficient for artificial insemination within 2-3 days. However, longer semen storage requires cryopreservation of boar semen. Freeze/thawing procedures induce sperm damage and induce reactive oxygen species in mammalian sperm and boar sperm seems to be more vulnerable for this than bull sperm. We developed a new method to detect reactive oxygen species induced damage at the level of the sperm plasma membrane in bull sperm. Lipid peroxidation in freshly stored and frozen/thawed sperm cells was assessed by mass spectrometric analysis of the main endogenous lipid classes, phosphatidylcholine and cholesterol and by fluorescence techniques using the lipid peroxidation reporter probe C11-BODIPY(581/591). Peroxidation as reported by the fluorescent probe, clearly corresponded with the presence of hydroxy- and hydroperoxyphosphatidylcholine in the sperm membranes, which are early stage products of lipid peroxidation. This allowed us, for the first time, to correlate endogenous lipid peroxidation with localization of this process in the living sperm cells. Cytoplasmatic droplets in incompletely matured sperm cells were intensely peroxidized. Furthermore, lipid peroxidation was particularly strong in the mid-piece and tail of frozen/thawed spermatozoa and significantly less intense in the sperm head. Induction of peroxidation in fresh sperm cells with the lipid soluble reactive oxygen species tert-butylhydroperoxide gave an even more pronounced effect, demonstrating antioxidant activity in the head of fresh sperm cells. Furthermore, we were able to show using the flow cytometer that spontaneous peroxidation was not a result of cell death, as only a pronounced subpopulation of living cells showed peroxidation after freeze-thawing. Although the method was established on bovine sperm, we discuss the importance of these assays for detecting lipid peroxidation in boar sperm cells.     

Generation of reactive oxygen species, lipid peroxidation, and human sperm function

Recent studies have demonstrated that human spermatozoa are capable of generating reactive oxygen species and that this activity is significantly accelerated in cases of defective sperm function. In view of the pivotal role played by lipid peroxidation in mediating free radical damage to cells, we have examined the relationships between reactive oxygen species production, lipid peroxidation, and the functional competence of human spermatozoa. Using malondialdehyde production in the presence of ferrous ion promoter as an index of lipid peroxidation, we have shown that lipid peroxidation is significantly accelerated in populations of defective spermatozoa exhibiting high levels of reactive oxygen species production or in normal cells stimulated to produce oxygen radicals by the ionophore, A23187. The functional consequences of lipid peroxidation included a dose-dependent reduction in the ability of human spermatozoa to exhibit sperm oocyte-fusion, which could be reversed by the inclusion of a chain-breaking antioxidant, alpha-tocopherol. Low levels of lipid peroxidation also had a slight enhancing effect on the generation of reactive oxygen species in response to ionophore, without influencing the steady-state activity. At higher levels of lipid peroxidation, both the basal level of reactive oxygen species production and the response to A23187 were significantly diminished. In contrast, lipid peroxidation had a highly significant, enhancing effect on the ability of human spermatozoa to bind to both homologous and heterologous zonae pellucidae via mechanisms that could again be reversed by alpha-tocopherol. These results are consistent with a causative role for lipid peroxidation in the etiology of defective sperm function and also suggest a possible physiological role for the reactive oxygen species generated by human spermatozoa in mediating sperm-zona interaction.     

Increased germ cell degeneration and reduced germ cell:Sertoli cell ratio in stallions with low sperm production

To determine the relationship between germ cell degeneration or germ cell:Sertoli cell ratio and daily sperm production, testes were obtained during the months of May to July (breeding season) and November to January (nonbreeding season) from adult (4 to 20-yr-old) stallions with either high (n = 15) or low (n = 15) sperm production. Serum was assayed for concentrations of LH, FSH and testosterone. Testes were assayed for testosterone content and for the number of elongated spermatids, after which parenchymal samples were prepared for histologic assessment. Using morphometric procedures, the types and numbers of spermatogonia, germ cells and Sertoli cells were determined. High sperm producing stallions had greater serum testosterone concentration, total intratesticular testosterone content, testicular parenchymal weight, seminiferous epithelial height, diameter of seminiferous tubules, numbers of A and B spermatogonia per testis, number of Sertoli cells per testis, and number of B spermatogonia, late primary spermatocytes, round spermatids and elongated spermatids per Sertoli cell than low sperm producing stallions (P < 0.05). The number of germ cells (total number of all spermatocytes and spermatids in Stage VIII tubules) accommodated by Sertoli cells was reduced in low sperm producing stallions (18.6 +/- 1.3 germ cells/Sertoli cell) compared with that of high sperm producing stallions (25.4 +/- 1.3 germ cells/Sertoli cell; P < 0.001). The conversion from (yield between) early to late primary spermatocytes and round to elongated spermatids was less efficient for the low sperm producing stallions (P < 0.05). Increased germ cell degeneration during early meiosis and spermiogenesis and reduced germ cell:Sertoli cell ratio was associated with low daily sperm production. These findings can be explained either by a compromised ability of the Sertoli cells to support germ cell division and/or maturation or the presence of defects in germ cells that predisposed them to degeneration.     

Cytological evaluation of the germ cell development strategy within the testis of the American alligator, Alligator mississippiensis

The cytological changes to germ cells were investigated within the seminiferous epithelium of the American alligator (Alligator mississippiensis). Testicular tissues were collected, embedded in plastic, sectioned on an ultramicrotome, and stained with the periodic acid–Schiff+ procedure followed by a haematoxylin counterstain. Alligators have a prenuptial pattern of germ cell development, where spermatogenesis begins in early spring and sperm is mature by the time mating begins in May. Consistent spatial relationships between germ cells are absent within the seminiferous epithelium of the alligator. Their germ cells progress through the phases of spermatogenesis as a single cohort, leading to one continuous spermiation event that occurs during their mating season (May–June). This temporal germ cell development is different from the consistent spatial development seen within seasonally breeding birds and mammals but is similar to the recently described germ cell development strategies of two other temperate breeding reptiles, the slider turtle and the European wall lizard. The germ cell development strategy shared by these three temperate reptiles representing three different taxa within the class Reptilia is reminiscent of the temporal strategy seen within the anamniotic testis. Thus, alligators and at least two other temperate reptiles exhibit primitive spermatogenic cycles within derived amniotic testes and may be consider intermediates in terms of testicular organization, which may have significance phylogenetically.     

Cadmium effects on sperm morphology and semenogelin with relates to increased ROS in infertile smokers: An in vitro and in silico approach

Smoking releases cadmium (Cd), the metal toxicant which causes an imbalance in reactive oxygen species level in seminal plasma. This imbalance is envisaged to impair the sperm DNA morphology and thereby result in male infertility. In order to correlate this association, we performed in vitro and in silico studies and evaluated the influence of reactive oxygen species imbalance on sperm morphology impairments due to smoking. The study included 76 infertile smokers, 72 infertile non-smokers, 68 fertile smokers and 74 fertile non-smokers (control). Semen samples were collected at regular intervals from all the subjects. Semen parameters were examined by computer assisted semen analysis, quantification of metal toxicant by atomic absorption spectrophotometer, assessment of antioxidants through enzymatic and non-enzymatic methods, diagnosis of reactive oxygen species by nitro blue tetrazolium method and Cd influence on sperm protein by in vitro and in silico methods. Our analysis revealed that the levels of cigarette toxicants in semen were high, accompanied by low levels of antioxidants in seminal plasma of infertile smoker subjects. In addition the investigation of Cd treated sperm cells through scanning electronic microscope showed the mid piece damage of spermatozoa. The dispersive X-ray analysis to identify the elemental composition further confirmed the presence of Cd. Finally, the in-silico analysis on semenogelin sequences revealed the D-H-D motif which represents a favourable binding site for Cd coordination. Our findings clearly indicated the influence of Cd on reactive oxygen species leading to impaired sperm morphology leading to male infertility.     

Reactive oxygen species signaling in primordial germ cell development in Drosophila embryos

REDOX mechanisms that induce biosynthesis of the reactive oxygen species (ROS) have attracted considerable attention due to both the deleterious and beneficial responses elicited by the reactive radical. In several organisms including Drosophila melanogaster, modulation of ROS activity is thought to be crucial for the maintenance of cell fates in developmental contexts. Interestingly, REDOX mechanisms have been shown to be involved in maintaining progenitor fate of stem cells as well as their proliferation and differentiation. Here, we have explored the possible functions of ROS during proper specification and developmental progression of embryonic primordial germ cells (PGCs). Indicating its potential involvement in these processes, ROS can be detected in the embryonic PGCs and the surrounding somatic cells from very early stages of embryogenesis. Using both “loss” and “gain” of function mutations in two different components of the REDOX pathway, we show that ROS levels are likely to be critical in maintaining germ cell behavior, including their directed migration. Altering the activity of a putative regulator of ROS also adversely influences the ability of PGCs to adhere to one another in cellular blastoderm embryos, suggesting potential involvement of this pathway in orchestrating different phases of germ cell migration.     

Reactive oxygen species generation and human spermatozoa: The balance of benefit and risk

Although the generation of reactive oxygen species is an activity normally associated with phagocytic leucocytes, mammalian spermatozoa were, in fact, the first cell type in which this activity was described. In recent years it has become apparent that spermatozoa are not the only nonphagocytic cells to exhibit a capacity for reactive oxygen species production, because this activity has been detected in a wide variety of different cells including fibroblasts, mesangial cells, oocytes, Leyding cells endothelial cells, thryroid cells, adipocytes, tumour cell and platelets. Since the capacity to generate reactive oxygen species is apparently so widespread, the risk-benefit equation for these potentially pernicious molecules becomes a matter of intese interest. In the case of human spermatozoa, the risk of manufacturing reactive oxygen metabolites is considerable because these cells are particularly vulnerable to lipid peroxidation. Indeed, there is now good evidence to indicate that oxygen radicals are involved in the initiation of peroxidative damage to the sperm plasma membrane, seen in many cases of male infertility. This risk is off-set by recent data suggesting that superoxide anions and hydrogen peroxide also participate in the induction of key biological events such as hyperactiavated motility and the acrosome reaction. Thus, human spermatozoa appear to use reactive oxygen species for a physiological purpose and have the difficult task of ensuring the balanced generation of these potentially harmful, but biologically important, modulators of cellular function.     

The impact and potential etiology of teratospermia in the domestic cat and its wild relatives

Teratospermia (production of >60% morphologically abnormal sperm/ejaculate) is relatively common among various species in the family Felidae, which is comprised of 37 species. Over two decades of research in this area have produced a significant understanding of the phenotypic expression, its impacts on sperm function and etiology. There is good evidence suggesting that a reduction in genetic diversity contributes to this phenomenon. Results to date demonstrate that spermatozoa from teratospermic donors are compromised in the ability to undergo capacitation and the acrosome reaction, penetrate the zona-pellucida, fertilize conspecific oocytes and survive cryopreservation. Recent studies also reveal abnormalities in chromatin integrity in sperm from teratospermic donors, which, interestingly, fails to impact fertilization or embryo development after intracytoplasmic sperm injection. Through planned inbreeding studies, we now have established that teratospermic cats also produce more spermatozoa by virtue of more sperm producing tissue, more germ cells per Sertoli cell and reduced germ cell loss during spermatogenesis. Overall, it now is clear that gain in sperm quantity is achieved at the expense of sperm quality, suggesting an extensive disruption of normal testicular function in teratospermic donors. Preliminary studies on testicular gene expression in teratospermic cats have also revealed abnormal expression patterns. These findings have markedly increased our understanding of testis biology in the teratospermic donor and reaffirm the value of cats, including wild species, as models for studying novel regulatory mechanisms controlling spermatogenesis and spermiogenesis.     

A hypothesis for the minimal overall structure of the mammalian plasma membrane redox system

Summary.  After a long period of frustration, many components of the mammalian plasma membrane redox system are now being identified at the molecular level. Some are apparently ubiquitous but are necessary only for a subset of electron donors or acceptors; some are present only in certain cell types; some appear to be associated with proton extrusion; some appear to be capable of superoxide production. The volume and variety of data now available have begun to allow the formulation of tentative models for the overall network of interactions of enzymes and substrates that together make up the plasma membrane redox system. Such a model is presented here. The structure discussed here is of the mammalian system, though parts of it may apply more or less accurately to fungal and plant cells too. Judging from the history of mitochondrial oxidative phosphorylation, it may be hoped that the development of models of the whole system – even if they undergo substantial revision thereafter – will markedly accelerate the pace of research in plasma membrane redox, by providing a coherent basis for the design of future experiments. Received May 4, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, United Kingdom. E-mail: ag24@gen.cam.ac.uk     

Essential role of neutrophils in germ cell-specific apoptosis following ischemia/reperfusion injury of the mouse testis

This study investigates the role of neutrophils in ischemia-induced aspermatogenesis in the mouse. Previous studies in the rat have demonstrated that ischemia-inducing testicular torsion followed by torsion repair and reperfusion resulted in germ cell-specific apoptosis. This was correlated with an increase in neutrophil adhesion to subtunical venules, an increase in reactive oxygen species, and increased expression of several apoptosis-associated molecules. In the present investigation, wild-type C57BL/6 mice were subjected to various degrees and duration of testicular torsion. A torsion of 720 degrees for 2 h caused disruption of the seminiferous epithelium and significantly reduced testis weight and daily sperm production. An immunohistochemical method specific for apoptotic nuclei indicated that these effects were due to germ cell-specific apoptosis. An increase in myeloperoxidase (MPO) activity and an increase in the number of neutrophils adhering to testicular subtunical venules after torsion repair/reperfusion demonstrated an increase in neutrophil recruitment to the testis. In contrast, E-selectin knockout mice and wild-type mice rendered neutropenic showed a significant decrease in neutrophil recruitment as evidenced by MPO activity and microscopic examination of subtunical venules. Importantly, germ cell-specific apoptosis was also reduced. Thus, germ cell-specific apoptosis is observed after ischemia/reperfusion of the murine testis, and this apoptosis is directly linked to the recruitment of neutrophils to subtunical venules. Endothelial cell adhesion molecules, particularly E-selectin, play an important role in mediating this pathology.     

Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing

In the present study, we provide evidence for the production of reactive oxygen species (ROS) during cryopreservation of bovine spermatozoa. Cooling and thawing of spermatozoa cause an increase in the generation of superoxide radicals. Although nitric oxide production remains unaltered during sperm cooling from 22-4 degrees C, a sudden burst of nitric oxide radicals is observed during thawing. Increase in lipid peroxidation levels have been observed in frozen/thawed spermatozoa and appears to be associated with a reduction in sperm membrane fluidity as detected by spin labeling studies. The data presented provide strong evidence that oxygen free radicals are produced during freezing and thawing of bovine spermatozoa and suggest that these reactive oxygen species may be a cause for the decrease in sperm function following cryopreservation. Mol. Reprod. Dev. 59: 451-458, 2001.     

Current status of sperm cryopreservation: why isn't it better?

Cryopreservation extends the availability of sperm for fertilization; however, the fertilizing potential of the frozen-thawed sperm is compromised because of alterations in the structure and physiology of the sperm cell. These alterations, characteristics of sperm capacitation, are present in the motile population and decrease sperm life-span, ability to interact with female tract, and fertilizing ability. The etiology of such alterations may represent a combination of factors, such as inherited fragility of the sperm cell to withstand the cryopreservation process and the semen dilution. Although the former is difficult to address, approaches that make-up for the dilution of seminal fluid may be sought. The aim of this work is to review aspects of sperm cryopreservation paralleled by events of capacitation and evaluate the possible roles of sperm membrane cholesterol, reactive oxygen species, and seminal plasma as mediators of cryopreservation effects on sperm function.     

The Amoroso Lecture. The human spermatozoon--a cell in crisis?

A great deal of evidence has accumulated in recent years to suggest that there has been a gradual increase in male reproductive pathology over the past 30-40 years, as evidenced by increased rates of testicular cancer and declining semen quality. The hypothesis is advanced that this phenomenon is causally related to the ability of male germ cells to generate reactive oxygen metabolites. When produced in low levels, such metabolites are thought to enhance sperm function by stimulating DNA compaction and promoting a redox-regulated cAMP-mediated pathway that is central to the induction of sperm capacitation. When produced in excessive amounts, the same metabolites stimulate DNA fragmentation and a loss of sperm function associated with peroxidative damage to the sperm plasma membrane. Free radical-induced mutations in the male germ line may also be involved in the aetiology of childhood cancer and recent increases in the incidence of seminoma. In light of these considerations, establishing the mechanisms for free radical generation by the male germ line and determining the factors that influence this activity are important objectives for future research in this area.     

Ultrastructure of the reproductive system of the black swamp snake (Seminatrix pygaea). V. The temporal germ cell development strategy of the testis

The germ cell development strategy during spermatogenesis was investigated in the black swamp snake (Seminatrix pygaea). Testicular tissues were collected, embedded in plastic, sectioned by ultramicrotome, and stained with methylene blue and basic fuchsin. Black swamp snakes have a postnuptial pattern of development, where spermatogenesis occurs from May to July and spermiation is completed by October. Though spatial relationships are seen between germ cells within the seminiferous epithelium during specific months, accumulation of spermatogonia and spermatocytes early in spermatogenesis and the depletion of spermatocytes and accumulation of spermatids late in spermatogenesis prevent consistent cellular associations. This temporal germ cell development within an amniotic testis is consistent with that seen in other recently studied temperate reptiles (slider turtle and wall lizard). These reptiles’ temporal development is more similar to the developmental strategy found in anamniotes than the spatial germ cell development that characterizes birds and mammals. Our findings also imply that a third germ cell development strategy may exist in temperate breeding reptiles. Because of the phylogenetic position of reptiles between anamniotes and other terrestrial amniotes, this common germ cell development strategy shared by temperate reptiles representing different orders may have significant implications as far as the evolution of sperm development within vertebrates.