Initiation and Development of Semen Production in the Guinea Pig

Transcription

1 Initiation and Development of Semen Production in the Guinea Pig M. FREUND, Ph.D. SEXUAL DEVELOPMENT and maturation of male animals may be studied by the use of a number of technics, such as the sacrifice at regular intervals of maturing animals with histological examination of testes and accessory organs, the observation of the onset and development of sexual behavior, or the. examination of ejaculated semen. In the past, the value of the semen examination as a measure of sexual development has been seriously limited by its dependence on the development of sexual behavior, since semen could not be collected until the animal was ready to mate-i.e. mount, intromit, and ejac~late, three distinct but interrelated activities that have been shown to develop at different rates in the guinea pig. 1 However, with the development of the technics of electroejaculation, it has become possible to obtain semen samples,from young males at an age earlier than that at which it could be collected by use of the artificial vagina. Electroejaculation has the advantage of avoiding the problem of libido and its rate of development, which might be quite different from the rate of development of spermatogenesis. Sayles collected semen from guinea pigs by applying three 10-sec. shocks 'of 6-30 v A. C., with a needle electrode at the base of the skull and a blunt electrode in the mouth. These stimuli resulted in violent whole-body nervous and muscular responses which included ejaculation. The coagulated ejaculates were weighed and examined for spermatozoa. The earliest appearance of sperm was at 54 days and the latest at 116 days and the weight of the ejaculate reached adult levels at 142 days. Recently, Oriol-Bosch and Parada used similar methods to collect semen by electroejaculation from guinea pigs and found that the pigs became apneic, had strong tonic muscular contractions and eversion of the anal mucosa; and then ejaculated. Artificial respiration was used to speed recovery from the procedure. They reported 116 ejaculations in 148 attempts at electroejaculation (72 per cent success). From the Department of Physiology and Pharmacology, New York Medical College, Flower and Fifth Avenue Hospitals, New York, N. Y. This investigation was supported by PHS RG-6099 and, in part, by PHS RG-6014 from the Division of General Medical Sciences, Public Health Service. 190

2 VoL. 13, No.2, 1962 SEMEN PRODUCTION 191 The development of more precise and less traumatic methods 7 for the electroejaculation of guinea pigs and for the liquefaction of the collected semen coagulum or "rodent vaginal plug," makes possible the study of important semen characteristics, including ejaculate volume, sperm concentration, and sperm motility, which could not be measured in the gelatinous, rubbery semen coagulum collected by earlier technics. The use of these newer methods of electroejaculation has resulted in successful electroejaculation in every case in this study. None of the undesirable side effects described in the earlier studies, i.e. cessation of breathing, massive tonic muscular contractions, eversion of the anal mucosa, etc., have been noted in our work, nor were other untoward effects on guinea pig growth, weight, or general health. A preliminary report on this work has been made. 8 METHODS Twenty-three male guinea pigs of the English breed (Rockland Farms), 3-4 weeks old, weighing gm. and born within a time span of 5 days, were housed in individual cages on wire grilles and were maintained on Purina guinea pig pellets, bread ad libitum, daily greens, and tap water. Each pig was electroejaculated, as previously described, 7 at biweekly intervals from the fourth through sixteenth weeks of life (one-half of the pigs collected one week and the other half the next week) and at weekly intervals from the seventeenth through twentieth weeks of life. Each semen specimen was collected by electroejaculation into a 5-ml graduate containing 1 mg. chymotrypsin in 1 ml. of phosphate buffer, after White, as previously described. 7 The chymotrypsin phosphate liquefied the semen coagulum produced upon ejaculation. Ejaculate volume was read to the nearest 0.1 ml., sperm concentration was determined by hemocytometer count, and the percentage of motile sperm was estimated in a warm stage at 39 C. under low power of the microscope. Tissue specimens from testes, coagulating gland, dorsal and lateral prostates, and seminal vesicles were fixed in Bouin's solution, sectioned at 5 fl, and stained with hematoxylin and eosin. Semen production RESULTS No sperm were found in any of the semen specimens collected from the 23 guinea pigs at 4-5 weeks of age. Ejaculate volume was small, ml., and only crystals, occasional epithelial cells, and debris were found in the semen smears.

3 192 FREUND FERTILITY & STERILITY Sperm appeared in the ejaculates of 2 of the pigs at 6 weeks of age and were observed to be nonmotile pin and amorphous heads. No normal sperm were found in the examination of these specimens. By 11 weeks of age, nonmotile sperm of normal appearance had been found in the ejaculates of half of the guinea pigs, although the cell concentrations were so low, i.e., less than one sperm per low-power field, that no attempt to count them was made. During the eleventh to thirteenth weeks, normal sperm began to appear in larger numbers and it became possible to express sperm concentrations in terms of the number of sperm per low-power field of the microscope ( LPF), which is the mean of the sperm counts made in 10 low-power fields. During the eleventh to seventeenth weeks, sperm concentration rapidly increased and, by the eighteenth week, it was possible to count the cells in a hemocytometer and to express sperm concentration as millions of sperm per milliliter (Table 1). Sperm concentration at 20 weeks of age had not reached the mean sperm production of mature guinea pig males when this study was terminated. The mean sperm concentration of the 12 specimens counted in the hemocytometer during the eighteenth to twentieth weeks of life in this study was TABLE 1. Semen Characteristics of the Developing Guinea Pig No. of Body Ejaculate Age animals weight volume Sperm Motilityt (wk.) remaining (gm.) (ml.) concentrationt (%) 4" TFq 0 7" TFC ';I'FC TFC TFC 0 II" TFC TFC /LPF /LPF /LPF 15 16" /LPF /LPF 10 18" X 106Jml X 106 /mi " X 106/ml. 30 *Ages at which animals were sacrificed for histological study. tin pigs having sperm at that age. :!:Too few cells to count; less than one per low-power field. Per low-power field of the microscope. ---

4 VoL. 13, No. 2, 1962 SEMEN PRODUCTION X 10 6 /ml., while the mean sperm concentration of 168 specimens from mature guinea pigs of the same strain, in another study, was X 10 6 /ml. (unpublished data). Evidently, these male guinea pigs are not fully sexually mature even at 20 weeks of age, although they have reached the average mature body weight for this strain ( gm.). It is general practice to begin to use male guinea pigs of this strain for natural breeding at about weeks of age and they have not been reported to be inferior to older breeders for this purpose. No influence of sperm concentration on male fertility is evident after the minimum (motile) cell concentration required for conception is present in the ejaculate. Although sperm were found in the ejaculates of 2 of the pigs at 6 weeks of age and in the ejaculates of one-half of the pigs at 11 weeks of age, it was not until the eighteenth week that sperm were found in the ejaculates of all of the pigs (Table 1). This large order of variability is probably a reflection of the large number of interacting factors involved in the initiation of semen production. The presence of sperm in the ejaculates of 2 of the pigs at 6 weeks indicates that spermatogenesis, at least in these 2 animals, must have started during or even before the first week of life, since the spermatogenic cycle plus the time required for the sperm to move through the epididymis and vas deferens has been shown to be about days in the rat,3 about days in the bull, 9 and about days in the guinea pig, in concurrent studies in this laboratory. The first observation of motile sperm was made in the ejaculate of one pig at 11 weeks and sperm motility was not found in the ejaculates of other pigs until the fifteenth week. Motile sperm were found in the ejaculates of all of the pigs during the nineteenth week of life. The onset of motile sperm production in this group of pigs was concentrated in the period weeks of age, except for the one pig which produced motile sperm during the eleventh week (Fig. 1 ). Thus, the period of onset of motility was much shorter ( weeks) than the period of onset of sperm production ( 6-18 weeks), although the periods overlapped. Invariably, in this study, the emission of motile sperm by each animal was preceded by one or more ejaculates containing only nonmotile sperm. Evidently, the first sperm emitted by the developing male guinea pig are nonmotile, and motile sperm appear only after some weeks have elapsed. The reasons for this delay in the appearance of motile sperm and the mechanisms by which sperm motility is initiated remain to be investigated. Testis development At 4 weeks of age, lumina were apparent in all of the seminiferous tubules examined and variable amounts of mitotic activity were found in tlte sections

5 194 FREUND FERTILITY & STERILITY from 5 animals. Mature spermatogonia were present in almost all of the tubules, but very few spermatocytes and no spermatids or sperm were found. No sperm were found in any of the ejaculates at this age. At 7 weeks of age, 4 animals were sacrificed. Testis sections from one pig OODY PER- WEIGHT CENT IGMJ ()0 BODY WEIGHT IN GM. )( PERCENT WITH SPERM IN EJACULATE PERCENT WITH MOTILE SPERM IN EJACULATE X AGE IN WEEKS Fig. 1. Spe1m production in the guinea pig in relation to age. (No. 37), which had produced occasional sperm at electroejaculation in the sixth week, revealed much mitotic activity, many spermatids, and some sperm but no sperm free in the tubule lumina. Testis sections from 3 pigs which had not produced any sperm during electroejaculation at 6 weeks, revealed only a few spermatocytes and no spermatids or sperm in 2 of the pigs (No. 50 and 59) and only a few spermatids and an occasional sperm with many tubules> without either sperm or spermatids in one (No. 63). At 11 weeks of age, 4 animals were sacrificed; 2 of which (No.?3 and 60) had 3-5 sperm per low-power field in their electroejaculates and 2 of which (No. 39 and 40) had no sperm in their ejaculates. Testis sections from pigs No. 53 and 60 showed great activity and many sperm in most of the lumina, with the testis presenting an almost mature appearance in all respects. Sections from pig No. 39 showed some sperm in the lumina of many tubules and

6 VoL. 13, No. 2, 1962 SEMEN PRODUCTION 195 spermatids in all of the tubules examined. There were no sperm and few spermatids and most of the tubules contained only spermatocytes and spermatogonia in the testis sections from No. 40. At 16 weeks of age, 4 animals were sacrificed. Testis sections from 3 pigs (No. 43, 51, and 52) which had produced sperm in their electroejaculates revealed an essentially mature picture, with masses of sperm in the lumina of many tubules. These sections were characterized by the great variability typical of mammalian testes, with tubules filled with masses of sperm next to tubules containing few or no sperm, but with many spermatids and other forms. The fourth pig (No. 64) presented a similar testis picture although this animal had not produced any sperm in 6 biweekly electroejaculations. Four animals were sacrificed at 18 weeks of age and 4 at 22 weeks. Sections from the testes presented a completely mature appearance with great masses of sperm in the lumina of most of the tubules. The observations of the histological studies of the testis sections and the examinations of the electroejaculated semen specimen were in close agreement. In almost every case where masses of sperm were visible in the lumina of the seminiferous tubules, there was at least one previous electroejaculate which contained sperm. The sole exception was pig No. 64, where the testis presented an essentially mature appearance upon histological examination, although no sperm had been observed in 6 electroejaculates collected at biweekly intervals. However, the average testis section assumed a mature histological appearance about 4-6 weeks before an average semen specimen containing a high concentration of sperm was electroejaculated. This may indicate that sperm migration into the epididymis and vas at a mature rate takes place in the guinea pig some weeks after spermatogenesis has reached the mature rate. Accessory gland development Perhaps the most striking features of the histological development of the seminal vesicles, coagulating gland, and dorsal and lateral prostates in the guinea pig are the early attainment of mature histological form, as reflected by cell size and appearance, and of gland function, as reflected by the amount and distribution of colloid and by the volume of ejaculated semen. The sections of seminal vesicle, at 4 weeks of age, contained many tall columnar epithelial cells with some limited areas of pseudostratification and were characterized by relatively small lumina filled with colloid. At 7 weeks of age, there was but little change, with larger lumina and more darkerstaining colloid being characteristic but with little or no change observed in cell type or height. At 11 weeks of age, the seminal vesicles attained their mature appearance and were characterized by very regular tall columnar

7 196 FREUND FERTILITY & STERILITY cells with basal nuclei, thrown into deep folds, surrounding large lumina filled with dark-staining colloid. Some areas of pseudostratification were observed in almost all of the sections studied. No further changes were found at 16, 18, or 22 weeks of age. The mature histological appearance of the seminal vesicle at 11 weeks of age is in accord with the observation that the mature level of semen production, in terms of ejaculate volume, is reached at that age (Table 1). The histological development of the coagulating gland was similar. At 4 weeks of age, the sections contained lumina filled with colloid, surrounded by low columnar cells with basal nuclei. Even at this age, there was considerable infolding of the epithelium into the lumina and the colloid stained a dark red. The changes at 7 weeks of age included some increase in size of the lumina and a greater amount of infolding of the epithelium, with the tall columnar type of cell being characteristic. At 11 weeks of age, the coagulating gland assumed the mature histological appearance with large alveoli filled with dark-staining colloid surrounded by highly folded tall columnar cells with some pseudostratified cells being observed in most of the sections. No further maturation changes were observed at 16, 18, or 22 weeks of age. The histological development of both the dorsal and the lateral prostates was similar and parallel to the development of the coagulating gland. At 4 weeks of age, the dominant cell types were columnar and pseudostratified columnar cells and the alveoli were relatively small but contained secretion. At 7 weeks of age, the columnar and pseudostratified columnar epithelial cells were taller and the epithelium had become highly folded into the alveolar lumen, which was full of colloid secretion. There was little change in the histological appearance of the dorsal and lateral prostates between the seventh and the eleventh weeks of age and these tissues seem to assume their mature histological form some time during this period. No further changes were observed at 16, 18, or 22 weeks of age. The histological observations of the testis and accessory glands in this study did not differ from the reports of others on maturing male guinea pigs. Therefore, the influence of regular biweekly electroejaculation on the histology of the developing testis and accessory glands is assumed to be negligible, if it exists at all, and no illustrations of the histological work are presented in this paper. In a previous study, 7 no effect of regular weekly electroejaculation on testis and accessory gland histology was found in mature guinea pigs. DISCUSSION This study was concerned with ( 1) establishing the time and manner of the initiation of semen production in the guinea pig; ( 2) following the de-

8 VoL.l3, No.2, 1962 SEMEN PRODUCTION 197 velopment of semen production before, during, and after puberty; and ( 3) correlating these data with the previously established histological changes during development. The age at first appearance of sperm (nonmotile) in the ejaculate varied widely, being observed in the ejaculates of 2 pigs at 6 weeks, of half the pigs at 11 weeks, and of all pigs at 18 weeks. These data are in very close accord with those of the study by Sayles, who found the first sperm at 54 days and sperm in all electroejaculates at 116 days in the guinea pig. Why the time of onset of semen production varies so widely in the guinea pig, and in other species as well, has not been established. Certainly this three-fold order of variability is much larger than the variability in time of onset of most other physiological functions in growing guinea pigs. The appearance of motile sperm in the ejaculate came several weeks after the first ejaculation of nonmotile sperm in each of the animals studied. The reason for this time lag before the onset of sperm motility is not known. It may be suggested that increasing levels of gonadotrophin andjor androgen, concomitant with increase in age, play a role in the onset of sperm motility. This will be considered in studies which are planned to investigate the effects of exogenous gonadotrophins on the development of semen production. The early appearance of sperm (nonmotile and, later, motile) in the guinea pig may be contrasted with the time of onset of sperm production in the bull, with which intensive studies have been made. Flipse et al., using the artificial vagina and a teaser female, found the first motile sperm in the bull ejaculate at 45 weeks, and Davies et al., using electroejaculation, reported the appearance of sperm at 9 months. It is of special interest that in the work of Davies et al., attempts to collect semen by electroejaculation from bulls less than 4 months old were unsuccessful and the first electroejaculates were obtained at 4J~ months of age. This may be compared to the present study where semen was collected by electroejaculation from guinea pigs at 4 weeks of age and with the work of Sayles, where an electroejaculate was obtained from a guinea pig as early as 21 days of age. Evidently, the ejaculation reflex itself, as measured by the response to electrical stimuli, develops at different rates in different species. The mean volume of electroejaculated semen reached 0.61 ml. at 11 weeks (Table 1) and there was no further significant increase in volume through the twentieth week. These data are not in agreement with those of Sayles, who reported that the weight of the coagulated electroejaculates reached adult levels at 142 days in the guinea pig. Since he did not attempt to liquefy the semen, he weighed the coagulated specimens and found that the average ejaculate at puberty weighed mg. (approximately ml.). This is a semen volume considerably lower than that reported in this study (Table

9 198 FREUND FERTILITY & STERILITY 1) and may be due to the difference in the methods used for electroejaculation. It is felt that the increase in volume of ejaculated semen during the first 11 weeks of life is a more useful measure of seminal vesicle functional development and maturation than is the use of seminal vesicle weight, as has been the case. Since, in the guinea pig, the largest part of seminal vesicle weight is made up of secretion, weight increase in itself is primarily a reflection of an increase in secretory activity rather than of organ growth. Sayles reported that the accessory organs of reproduction in the guinea pig gradually increased in average weight up to 30 days and then showed a marked increase in average weight. This type of weight increase data defies analysis since the increase in gland tissue weight, the increase in secretion rate, the cumulative storage of secretion, and the variability among animals are confounded. However, when semen is regularly collected from the same animals by electroejaculation, the problem of cumulative storage of the secretion is avoided and the ejaculate volume is, therefore, a direct reflection of the secretory activity of the accessory glands of reproduction (primarily the seminal vesicle). Furthermore, the data used in this experiment are repeated biweekly measurements made on the same animals while, of necessity, the studies which use changes in accessory gland weights as criteria of maturation must sacrifice the animals and, therefore, must compare only group means. Levey and Szego studied fructose production by the seminal vesicle of the immature guinea pig and found that, at 20 days of age, the concentration of fructose in the seminal vesicle secretion was at the adult level. Thev concluded from their observation, and the report of Sayles of first spen~ at 54 days, that the seminal vesicle in the guinea pig responds to the start of endogenous androgen secretion before the testis tubules do. However, they did not consider that days are required for spermatogenesis plus sperm migration through the male genital tract. If 48 days are added to the original 20, the time estimates for the effects of the start of endogenous androgen secretion on the initiation of spermatogenesis come well within the range for the first ejaculation of sperm reported in this study ( 6-18 weeks) and by Sayles ( days). These data are in accord with work with the boar, 2 where it was found that ejaculate volume, sperm concentration, and sperm motilitv showed progressive increases with increase in age, over the period of days of life, and with the bull, 6 where it was found that similar increases took place in volume, concentration, and motility during the sixtieth to ninetieth weeks of life. In both these studies, however, improvement in semen quality paralleled increase in age and also in body weight, while in this work the

10 VoL. 13, l\"o. 2, 1962 SEMEN PRODUCTION 199 guinea pigs reached their mature body weight at 20 weeks but had not yet reached their mature level of sperm production (Fig. 1). This difference may be explained, at least in part, by the fact that the ejaculates in the boar and bull studies were collected by artificial vagina and, therefore, part of the improvement in semen quality with increase in age might be associated with increase in libido with increase in age as well as with the period of time required by the male to become completely conditioned to serve the artificial vagina, using a dummy (in the case of the boar) or a teaser female (in the case of the bull). The technic of electroejaculation makes it possible to avoid the problem of accounting for the influence of these other variables in such studies. If puberty may be defined as the age at which the reproductive organs become functionally operative and secondary sex characters develop, 5 electroejaculation may be used as a technic that offers a practical approach to the problem of identifying the age of puberty in the male laboratory animal. It is proposed that the age of puberty of a male animal be set as the earliest age at which semen containing motile spermatozoa may be collected by electroejaculation from that animal. The age of puberty among the males of any given species and strain would then be the mean age at which motile sperm may be electroejaculated- the eighteenth week of life in the guinea pig. Electroejaculation is specified since guinea pigs/ bulls, 4 and boars, 2 have been shown to produce motile sperm before they will mount, intromit, and ejaculate-three interrelated processes that are, nevertheless, distinct in terms of rate of development in the young male. This type of measurement of puberty should prove to be more objective and reproducible than the older and relatively subjective method of observation of the change in body form or of the development of sexual activity. Furthermore, this type of measurement does not involve the sacrifice of the individual animal as is the case in the use of testis and accessory gland for tissue-weight, histological, or biochemical determinations of puberty. It is also proposed that the age of sexual maturity of a male animal be set as the age at which semen containing the adult concentration of motile sperm for that species and strain may be collected by electroejaculation. These proposed measures sharply differentiate, for the first time, between puberty and sexual maturity in a laboratory animal, since in the guinea pig several weeks elapse from the age at which the first motile sperm may be collected until the adult specimen is collected. It is felt that the objectivity and reproducibility of the proposed measurements of puberty and of sexual maturity will make it much more feasible to plan studies on the physiological basis for sexual development, puberty, and maturity, and on the factors affecting them.

11 200 FREUND FERTILITY & STERILITY The sperm concentrations of the electroejaculated semen specimens in this study accurately reflected the histological status of the testis. The semen specimens also yielded other measurements, sperm motility and ejaculate volume, which reflect sexual development and maturation and which are not available in histological studies. A major advantage of this type of study is the repeated collection of semen from the same animal so that maturation and/or treatment effects may be measured, using each animal as its own control. The advantages of electroejaculation as a method of obtaining repeated semen specimens, at regular intervals, make it the method of choice in studies of puberty and sexual maturity in the male guinea pig. SUMMARY Semen was regularly collected by electroejaculation from 23 guinea pigs, during the fourth to twentieth weeks of life. Sperm appeared in the ejaculates of 2 pigs at 6 weeks, of half the pigs at 11 weeks, and of all pigs at 18 weeks. Sperm concentration had not reached mature levels at 20 weeks of age. Sperm motility was first observed at 11 weeks in the ejaculate of one pig and appeared during the period of the fifteenth to nineteenth weeks in the ejaculates of the other pigs. Mean ejaculate volume increased from 0.06 ~I. at 4 weeks to 0.61 mi. at 11 weeks and there was no further significant increase in volume through the twentieth week. There was close agreement between the results of histological studies of testis sections and those of examinations of the electroejaculated semen specimens. It is proposed that an objective and reproducible measure of the age of puberty of a male animal may be set as the earliest age at which semen containing motile spermatozoa may be collected by electroejaculation from that animal. Using this measure, the age of puberty of this strain of guinea pigs has been determined to be at the eighteenth week of life: Dept. of Physiology and Pharmacologtj New York Medical College Fifth Ave. and 106 St. New York 29, N.Y. REFERENCES 1. ANTUFF, H. R., and YoUNG, W. C. Internal secretory capacity of the abdominal testisin the guinea pig. Endocrinology 61:121, BANE, A. Some results of studies on the semen of young boars and on artificial insemination in pigs. Ann. de Zootechnie Supp.: 15, CASARETI', A. P., and CASARETT, G. W. Histological investigations of mechanisms of x-ray effects on spermatogenesis in the rat. U. Rochester A. E. Prof. Report UR-496, DAVIES, D. V., MANN, T., and RowsoN, L. E. A. Effect of nutrition on the onset of male"

12 VoL.13, No.2, 1962 SEMEN PRODUCTION 201 sex hormone activity and sperm formation in monozygous bull calves. Proc. Royal Soc., B 147:332, Dorland's IUustratei/, Medical Dictionary. Saunders, Philadelphia, FLIPSE, R. J., ALMQUIST, J. 0., and JoHNSON, P. E. Effect of proteins of plant and animal origin on the growth, reproductive development and semen production of young dairy bull. ]. Dairy Sci. 89:60, FREUND, M. Collection and liquefaction of guinea pig semen. Proc. Soc. Exper. Biol. & Med. 98:538, FREUND, M. Development of semen production in the guinea pig. Fed. Proc. 19(1): 371, KoEFOED-JoHNSON, H. H. Influence of ejaculation frequency on the time required for sperm formation and epididymal passage in the bull. Nature 185:49, LEVEY, H. A., and SZEGO, C. M. The effect of androgens on fructose production by the sex accessories of male guinea pigs and rats. Endocrinology 56:404, OruoL-BosCH, A., and PARADA, J. Fructose im ejaculat. Endokrinologie 89:14, SAYLES, E. D. Postnatal development of reproductive system in male guinea pigs and its relation to testis hormone secretion. Physiol. Zool. 12:256, WmTE, I. G. The effect of some seminal constituents and related substances on diluted mammalian spermatozoa. Australian]. Biol. Sci. 7:379, Fourth World Congress on Infertility The Fourth World Congress on Infertility, sponsored by the International Fertility Association, will be held at the Hotel Copocabana, Rio de Janiero, Aug. 8-15, For further information address DR. MAXWELL RoLAND, Secretary, st Road, Forest Hills 75, N.Y., U.S. A.