Nally Situated Testes and There Was No Pampiniform Plexus While in the Species with Scrotal Testes, It Was Coiled in the Region of the Plexus

ASPECTS OF SPERM PRODUCTION IN SOME EAST AFRICAN MAMMALS

T. D. GLOVER Unit of Reproductive Biology, University of Liverpool, P.O. Box 147, Liverpool L69 3BX (Received 1st September 1972) Summary. A comparison of certain features of the testis and epididymis has been made in five species of East African mammals, two of which, the rock hyrax and elephant, have abdominally situated testes. The artery to the testes was straight in the species with abdomi- nally situated testes and there was no pampiniform plexus while in the species with scrotal testes, it was coiled in the region of the plexus. It is suggested that where the testicular artery is coiled, the testes should be regarded as basically scrotal, even if they are usually found in the abdomen post mortem. A striking increase in blood flow in the testis of the rock hyrax during sexual activity suggests that the simpler arterial pattern of the testis in testicond mammals allows a greater variation in blood flow than the more complicated arterial design associated with scrotal testes. Characteristic signs of sperm maturation occur in the epididymis of testicond mammals in contrast to the situation in artificial cryptor- chidism, where normal epididymal function is completely disrupted. It is suggested that epididymal function, as well as spermatogenesis, has become modified during evolution. Evidence is given that a need for prolonged survival of spermatozoa in the mesonephric duct might have been an important primary factor in the caudal migration of gonads into a scrotum.

INTRODUCTION In this paper, reference is made mainly to the testis and epididymal contents of the giraffe, Giraffa, eland, Taurotragus, steinbok, Raphicerus, jumping hare, Pedetes, rock hyrax or dassie, Procavia and Heterohyrax, and elephant, Loxodonta, because examples of all these genera are found in equatorial Africa and thus have several features of environment in common. In the first four genera, the testes are situated in a scrotum. In the hyrax and the elephant, they are permanently and normally in the abdomen (mammalian testiconda). The jumping hare has a scrotal pouch but the testes have been described as being abdominal (Coe, 1969). In mammals in which the testes and epididymides are in a scrotum, the production of spermatozoa comprises spermacytogenesis, involving cell d 45 Downloaded from Bioscientifica.com at 09/29/2021 09:58:43AM via free access 46 T. D. Glover division, and spermateliosis, involving no cell division. The process of sperm maturation, therefore, begins in the testis but is continued in the epididymis long after the spermatozoa leave the testis. It is well known that in scrotal mammals, spermatogenic activity of the testis is highly susceptible to elevated temperature. It has also been shown that maturation and survival of sper¬ matozoa in the epididymis is extremely sensitive to increased temperature. In artificial cryptorchidism, for instance, epididymal spermatozoa show a rapid loss of function and soon degenerate (Glover, 1960; Cummins & Glover, 1970). In the mammalian testiconda, therefore, the question arises of how spermato¬ genesis proceeds at abdominal temperatures, and how spermatozoa can mature and survive in an epididymis that is permanently in the abdomen. Harrison (1949) pointed out the unusual arterial architecture of scrotally situated testes, and there is evidence to suggest that this might be a disadvantage to the organs in adapting to increased temperature (Setchell & Waites, 1970). For this reason, the course and distribution of the testicular artery was examined in the present work, particular attention being paid to animals with abdominal testes. Certain distinct changes are known to typify sperm maturation in the epididymis, and in mammals with scrotal testes, the tail of the epididymis is recognized as an area which is conducive to prolonged survival of mature spermatozoa. Although reference has been made to these events in the hyrax and the elephant (Glover, 1968; Glover & Sale, 1968), there is little information about them in African mammals generally. It was, therefore, decided to examine spermatozoa from different levels of the epididymis in the different species.

MATERIALS AND METHODS Material was collected from two eland bulls, two steinbok, three giraffe, eight rock hyraxes and three elephants in Kenya. Further investigations were made on rock hyrax from the Transvaal, South Africa, and on one elephant shot on the banks of the Crocodile River bordering the Kruger National Park, near Komatipoort. Examination of the testicular artery was carried out in each case according to the method of Harrison (1951), using an injection of radio-opaque material and the subsequent production of an arteriograph. Micropaque (barium sulphate, 100% w/v, Damancy & Co., Ltd) was used for direct injection into the testicular artery of larger species and into the thoracic or abdominal aorta of smaller species. The injection was always made slowly, either by using a sim¬ ple record syringe or by gravity feed, according to circumstances. The giraffe was the only species in which it was found necessary to flush out the artery with saline before introducing the injection fluid. The particle size of the Micropaque is such that it does not pass through capillaries and thus it only fills the arterial side of the vasculature. After injection, the testes were removed and stored at a temperature of 0° C, or lower, before X-rays were taken. For a thorough examination of material, it is difficult to perform all the required procedures on the same animal. Hence, spermatozoa were usually

Downloaded from Bioscientifica.com at 09/29/2021 09:58:43AM via free access Sperm production in some East African mammals 47 taken from separate fresh specimens which were dissected out and examined as soon as possible after the death of the animal. Spermatozoa were collected by incising the epididymis at different levels (Text-fig. 1) and gently squeezing out the spermatozoa onto a microscope slide. The first sample was quickly examined under the microscope for motility. If the spermatozoa were immotile, the sample was slightly diluted with fructose Ringer phosphate solution (Mann, 1964). No attempt was made to quantify motility; a subjective assessment of

Text-fig. 1. Homologous areas of the epididymis in a scrotal mammal (left) and a testicond mammal (right) from which spermatozoa were withdrawn for examination. 1 = caput epididymidis; 2 = cauda epididymidis.

'good', 'medium' or 'nil' was made. When this examination was complete, a further sample from the same area was taken, mixed with one drop of aqueous nigrosin eosin on a microscope slide, smeared onto the same slide and a dup¬ licate smear made on another slide from the residual spermatozoa-stain mixture on the cover slip. Each was allowed to dry in air before being mounted in DPX. Later, 100 spermatozoa were counted on each slide for an estimate of mor¬ phological features. Further smears were made for staining with Papanicolaou's stain (Papanicolaou, 1942) for confirmation of the morphological picture.

Downloaded from Bioscientifica.com at 09/29/2021 09:58:43AM via free access 48 T. D. Glover Since it was not possible to test the fertilizing capacity of the spermatozoa, a morphological indicator of sperm maturation was used in addition to the observation ofmotility. The criterion taken was the migration of the cytoplasmic droplet to the end of the mid-piece (Lagerlöf, 1934; Merton, 1939). Contraction of the acrosome round the nucleus as seen in maturing rabbit spermatozoa (Bedford, 1965) was unreliable as a guide to sperm maturation in this instance because conditions were conducive to membrane damage and any abnormal swelling of the acrosomes would make quantitative interpretations difficult. Measurements of sperm size were made with the use of an ordinary eyepiece micrometer gauge. Text-figure 1 illustrates the levels of the mesonephric duct from which sperma¬ tozoa were withdrawn in both scrotal and testicond species. The levels in each type of mammal are based on homologues suggested earlier (see Glover, 1968; Glover & Sale, 1968). RESULTS The course and distribution of the testicular artery In the eland and steinbok, the testicular artery forms a helix in the region of the pampiniform plexus and runs onto the surface of the testis underneath the tunica albugínea. Here, it divides dichotomously into two main arteries, the branches of which run into and supply the testis parenchyma. This arterial pattern is essentially the same as that in domesticated ruminants and is again to be seen in the testis of the giraffe. In the giraffe, however, the artery is more convoluted in the region of the pampiniform plexus and the coils extend onto the surface of the testis (PI. 1, Fig. 1) before the artery continues in its course under the tunica albugínea. In the jumping hare, the testicular artery is also coiled before reaching the testis. There is a distinct pampiniform plexus of veins and the artery courses tortuously under the tunica albugínea in a similar but more exaggerated fashion than that of the rat (PI. 1, Fig. 2). In contrast to these patterns, the testicular artery of the hyrax is straight (Harrison, 1958), although it may show a slight kink in some animals (PI. 1, Fig. 3). The artery does not run far on the surface of the testis but divides into two major arteries whose branches enter the testis directly from one side only. The left testicular artery arises from the inferior renal artery, there being three arteries to each kidney. The male rock hyrax is a seasonal breeder, and specimens were taken from both sexually active and sexually quiescent animals (Glover & Sale, 1968). Differences in the size of the testes between these two are often quite remarkable (Millar & Glover, 1970) and it was observed that, in sexually active animals, the testicular artery and its branches were much better filled with Micropaque than in sexually quiescent animals. It is thus evident that in sexual activity the blood vessels dilate and the testis becomes suffused with blood. Markedly distended veins filled with blood lend further support to this. Characteristics of epididymal spermatozoa A considerable difference was noted in the length and breadth of sperm heads

Downloaded from Bioscientifica.com at 09/29/2021 09:58:43AM via free access PLATE 1

Fig. 1. Arteriograph showing the arterial supply to the testis of a giraffe. Fig. 2. Arteriograph showing the arterial supply to the testis of a jumping hare. Fig. 3. Arteriograph showing the arterial supply to the testis of a rock hyrax. A = aorta; R = renal arteries; = testicular (internal spermatic) artery. Fig. 4. Arteriograph showing the straight testicular artery of an elephant.

(Facing p. 48)

Downloaded from Bioscientifica.com at 09/29/2021 09:58:43AM via free access Sperm production in some East African mammals 49 in the different species examined, as well as in the overall length of the sper¬ matozoa, in the shape of sperm heads and in the length of the mid-pieces. Table 1 indicates that in all the species, spermatozoa acquire the capacity for motility as they move through the mesonephric duct, and that during this time the cytoplasmic droplet migrates towards the end of the mid-piece. During epididymal transit, the acrosome appears to contract down over the nucleus of the spermatozoon, but this could be an artifact and needs to be checked with the electron microscope. It is to be emphasized, however, that signs of matura¬ tion in the spermatozoa are just as evident when the excurrent ducts are in the abdominal and pelvic cavities as when they are in a scrotum. Table 1. Incidence of spermatozoa with proximal cytoplasmic droplets and the moti¬ lity of samples collected from two levels of the epididymis in four species of African mammals

Level of mesonephric duct* Position No. of 1 of animals Species testes Droplets Droplets (%) with Motility (%) »Mi Motility ranges^ ranges^ Scrotal Giraffe 82 (75 to 91) Medium 1 (nil) Good Eland 74 (69 to 78) Medium 3 (1 to 5) Good Abdominal Elephant J 41 (39 to 43) Nil 12 (nil) Medium Hyrax 51 (42 to 60) Nil 1 (0 to 2) Good (sexually active)

* See Text-fig. 1. t Mean percentage from duplicate smears. % With fructose Ringer phosphate added (see text).

In all three elephants examined, there was little or no motility in sperm samples taken from any level of the mesonephric duct, but in one elephant it was found that sperm motility could be stimulated by the addition of fructose Ringer phosphate. When this was done, clear differences in the activity of immature and mature spermatozoa became apparent (see Table 1). In this context, therefore, the elephant would not seem to be basically different from other testicond mammals.

DISCUSSION In this work, arteriographs suggest that convolution of the testicular artery has evolved in association with descent of the testes. Taxonomically, it is reasonable to believe that abdominal retention of the testes and a corresponding straight testicular artery represent a relatively primitive condition and that differences in the degree of convolution of the artery are simply the result of evolutionary divergence. Harrison (1949) and Harrison & Weiner (1949) contended that the length of the testicular artery is related to the degree of cooling of the incoming blood, although this might depend partly on the ambient temperature. In this connection, it is interesting that, in the giraffe, the artery is excessively

Downloaded from Bioscientifica.com at 09/29/2021 09:58:43AM via free access 50 T. D. Glover coiled, since the animal is a tall ruminant and is regularly exposed to the sun for long periods. The testicular artery of the jumping hare is less coiled in the region of the pampiniform plexus, but coiling in the artery indicates that the testes in this species are essentially scrotal. When the animal is in a relaxed condition, the testes may be seen in the scrotum; it is only after death that they are almost always to be found in the abdomen. For this reason, they have erroneously been described as abdominal (Coe, 1969). It is possible, of course, that the jumping hare periodically withdraws its testes into the abdomen, but as with some lagomorphs which also show periodic withdrawal (Asdell, 1964), large inguinal canals and a coiled testicular artery may be taken as indicating that descent of the testes has at some time occurred during the animal's evolution. There is nothing more than this to suggest that a coiled testicular artery, which continues its course superficially under the tunica albugínea, is related to the susceptibility of the germinal epithelium to fluctuations in temperature, though the straightness of the artery in testicond mammals (PI. 1, Fig. 4) does suggest that abdominal testes might function rather differently from scrotally situated ones. The arterial design in the testes of testicond mammals would seem to allow a greater variation in blood flow, and in the hyrax signs of increased flow in the testes during sexual activity are striking. The increase in blood flow is interesting in relation to the animal's hormonal status at this time, when a high level of circulating gonadotrophins might be anticipated. In scrotally situated testes, there is evidence of considerable resistance to changes in blood flow (Glover, 1968; Setchell, 1970). It seems reasonable to conclude, therefore, that coiling of the artery in the region of the pampiniform plexus has been associated with testicular descent and that it might have been accompanied by speciali¬ zation of spermatogenic tissue as an adaptation to the unusual environment in a scrotum. Spermatogenesis in abdominal testes might not need an especially low temperature, and might even be stimulated when the temperature is increased. It has been shown that a high temperature stimulates spermatogenesis in the common frog, Rana temporaria, by accelerating the FSH activity of the pituitary (Van Oordt & Lofts, 1963) and the relationship of temperature and the hor¬ monal control of the testes in mammals bears closer examination. By contrast, development of spermatogenesis in young birds appears to be speeded up at temperatures lower than the abdominal temperature, but the process does not seem to favour very low temperatures (Williams, 1958), and there is no direct evidence that spermatogenesis in birds is hypersensitive to elevated tempera¬ tures. The possibility could be tested, but the fact that body temperature in birds is higher than that of mammals suggests that there is considerable variation between vertebrate species in the sensitivity of their testes to high temperature. This is certainly worth bearing in mind in the case of man, where the testes may have become partially adapted to increased temperature. There are marked differences in the dimensions of epididymal sperm heads in different species. It is probable, though, that the DNA content is relatively constant in mammalian spermatozoa and that apparent species variations in size mainly reflect differences in shape and thickness.

Downloaded from Bioscientifica.com at 09/29/2021 09:58:43AM via free access Sperm production in some East African mammals 51 In all the species examined here, however, spermatozoa acquired the capacity for motility as they traversed the mesonephric duct, and the cytoplasmic droplet migrated to the end ofthe mid-piece. These changes were as obvious in testicond mammals as in those with scrotal testes, and are of interest in view of the fact that sperm survival in the epididymis is severely curtailed in artificially cryptorchid animals. It seems, therefore, that the function of the epididymis has been modified in conjunction with its descent into a scrotum, neces¬ sitating a relatively stable temperature for sperm maturation. This can be provided by the scrotum in most mammals, but in testicond mammals, thermo¬ régulation is generally poor (Wislocki, 1949). In the rock hyrax, fluctuations in body temperature must be considerable (Taylor & Sale, 1969) as a result of the animal's normal habitat. Even if its body temperature is rather lower than that of most eutherian mammals, it would not adequately explain how its testes are able to function in the abdomen or its spermatozoa are able to mature in an abdominal epididymis when the ambient temperature is so variable. It certainly seems that the extreme sensitivity of testes to temperature fluctuation has occurred in association with the evolutionary development of homeothermy. Elephants, however, are out in the sun for most of the day and their body temperature has been shown to remain within average limits (Short, Mann & Hay, 1967). Nevertheless, more information on diurnal fluc¬ tuations in both body temperature and spermatogenesis in a wide variety of testicond mammals would be useful. The survival of mature spermatozoa in the cauda epididymidis has been shown in rabbits to be dependent upon maintenance of the temperature within a fairly narrow range below that of the abdomen (Chang, 1943; Glover, 1958). In testicond mammals, however, the comparable region of the mesonephric duct lies relatively close to the surface of the body (Glover & Nicander, 1971). In the rock hyrax and the elephant, this part of the duct is distended and whiter than the rest of the duct, and it seems justifiable to regard it, like the cauda epididymidis, as a region ofsperm storage. Spermatozoa probably move through it constantly, as they do through the cauda epididymidis, but the rate of transport must be much slower than in other regions, and the lumen is dis¬ tended and packed with spermatozoa. In sexually active animals, the contents of the duct at this level are under pressure and tend to flatten the surrounding epithelium. These general features appear to typify the 'true testiconda', for they are also seen in a number of insectivores such as Elephantulus, Petrodromus and Rhyncocyon (T. D. Glover, personal observations). If this interpretation of homologous areas is correct, and the contributory evidence is now substantial, the ductus deferens in testicond mammals will be represented only by a short terminal length of the mesonephric duct. From recent work (Glover & Nicander, 1971), it appears that, from a phylogenetic point of view, the sperm store in mammals has migrated towards the surface of the body and even left the confines of the abdomen before the testes. Testicular descent may well have been secondary to descent of the sperm store. At least, it would seem that during the course of evolution, the need for prolonged survival of spermatozoa in the mesonephric duct may have been a factor involved in the caudal migration of the gonads and their ducts.

Downloaded from Bioscientifica.com at 09/29/2021 09:58:43AM via free access 52 T. D. Glover

ACKNOWLEDGMENTS This work was aided by grants from the Ford Foundation and the Royal Society. My thanks are due to Dr W. Hopkirk of Nairobi for the arteriographs, to Dr R. Laws, Professor J. D. Skinner and Dr J. B. Sale for their help and interest in the work, to Mr T. Williams for technical assistance, and to Mrs J. Kelly for typing the manuscript. My thanks are also due to the authorities of the Kenya Game Development Board and the Kruger National Park, and to Professor R. G. Harrison for his helpful criticism of the manuscript.

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Downloaded from Bioscientifica.com at 09/29/2021 09:58:43AM via free access