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Transport of Spermatozoa Through the Female Genital Tract of Hamsters
Transport of Spermatozoa Through the Female Genital Tract of Hamsters Herbert S. Yamanaka, B.S., and A. L. Soderwall, Ph.D.
THE
on the rate of sperm transport through the female reproductive tracts in different species of mammals reveals considerable variation in findings. With improvement of technics for such studies, more recent reports have shown that less time is required for spermatozoa to traverse the female reproductive tract than was previously thought. Beschlevnov1 found that spermatozoa in the cow enter the uterus in less than 40 minutes after ejaculation and reach the ovarian end of the uterus in 4 hours. On the other hand, VanDemark and Moeller7 observed spermatozoa at the ovarian end of the fallopian tube in the cow in less than 3 minutes after artificial insemination to the midcervical region. In studies with the albino rat, Blandau and Monet noted spermatozoa in the uterine segments of the oviducts within 15 minutes after ejaculation in 21 per cent of the females; in one hour sperm were observed throughout the oviducts of all the mated rats. The work by VanDemark and Moeller7 indicates that the rate of spermatozoan transport is much greater than would be expected if sperm motility were the only factor involved in their movement within the female reproductive tract. The investigation reported here was carried out to determine the time required for spermatozoa to reach various levels of the female genital tract in the hamster (Mesocricetus auratus), and the mechanism of their transport. LITERATURE
From the Department of Biology, University of Oregon, Eugene, Ore. This study was aided by grants from the U. S. Public Health Services (No. 4213), and Faculty Research Committee of the University of Oregon. 470
Vol. 11, No.5, 1960
SPERM TRANSPORT
471
MATERIALS AND METHODS The data in this study were obtained from matings involving 85 female hamsters ranging in age from 2 to 21 months and 13 male hamsters of similar age range, used for matings. The animals were divided into 2 age groups: the young, consisting of animals ranging in age from 2 to 12 months, and the old, from 13 to 21 months. From these, 4 breeding groups were established (Table 1). Females in heat were placed in breeding boxes with vigorous males, as described by SoderwalL 6 The male hamster will ordinarily mount a female in heat a number of times before ejaculation. In order to observe the exact time of ejaculation, the male was allowed to mount a teaser female 10 times. During this period an experimental female, in heat, was placed in an adjacent box and after the tenth mounting the teased male was placed with the female. The exact moment of ejaculation was timed with a stop watch. The mated female was killed by a blow on the back of the head at intervals ranging from 1 to 2~~ minutes following ejaculation. The abdomen was opened immediately and the genital tract exposed. Thin hemostats were placed at the cornual-cervical junction (proximal third), midcornual (mid third), distal-cornual (distal third), and ampullary portion of the uterine tube (Fig. 1). The total elapsed time for this procedure was approximately 15 seconds. The cornual segments were isolated and the contents of the lumen were squeezed onto a clean glass slide and diluted 1: 200 parts with Locke's
Section Third
Fig. 1. Diagrammatic view of female hamster reproductive tract. Dashes indicate location of hemostats.
472
Fertility & Sterility
YAMANAKA & SODERWALL
solution in a blood pipet; counting was done with a hemocytometer. The ampullae were rinsed thoroughly in Locke's solution and observed for the presence of spermatozoa. OBSERVATIONS Table 1 summarizes the essential data and shows the distribution of spermatozoa within the reproductive tract in females killed on an average of approximately 2 minutes after ejaculation. TABLE 1.
Sperm Transport Times in Female Reproductive Tract Cornual segments with sperm
Group
Average No. age females (months) used
Proximal L. R. %
Middle L.
R.
%
L.
Distal
R. %
Average Average Ampulla time time (sec.).' L. R. % (sec.)t
1 Young female
8
X Young male
27
27 27 100 24 25 90 16 15 57
105
8 7 26
115
20
20 19
6 35
120
3 1 10
155
18
18 18 100 15 15 83
9 11 55
115
5 5 22
127
20
19 18
0
120
o
9
2 Old female
15
X Young male
98
12 13 62.3 8
10
3 Young female
9
X Old male
15
4 Old female
18
X Old male
92.5 7
5 30
0
0
0
0
17
• Average time of sacrifice for whole grouP.
t Average time of sacrifice tor animals in which sperm were found in the ampulla.
Group 1.
In matings between males and females of 8 to 9 months of age, all of the 27 animals sacrificed showed spermatozoa in all parts of the cornua at the end of 2 minutes. Fifty-four cornua were examined at an average of 105 seconds; 100 per cent showed spermatozoa at the proximal end, 90 per cent at the middle portion, and 67 per cent at the distal end. Twenty-six per cent of the 54 ovarian segments of the cornua contained 1-200 spermatozoa. The average time of examination in these cases was 115 seconds. However, in one instance, 4 spermatozoa were observed in this region after H minutes. Group 2. Twenty old females were mated with young males and killed at an average time of 120 seconds after ejaculation. Spermatozoa were present in 98, 62, and 35 per cent in the proximal, middle, and distal sections
Vol. II, No.5, 1960
SPERM TlIANSPORT
473
of the uterine cornua, respectively. In the ampullary region, 10 per cent of the sections were infiltrated by spermatozoa; the average time in these instances was 155 seconds. Group 3. In the 18 pairs of cornua from young females bred with old males, the results were much like those in Group 1. At the average sacrifice time of 115 seconds, 100, 83, and 55 per cent showed spermatozoa at the proximal, mid, and distal sections, respectively. In the ampullary region, 22 per cent showed positive results at an average of 127 seconds. Group 4. Twenty old females were mated with old males and the females sacrificed at an average of 2 minutes after ejaculation. Spermatozoa were not noted in the ampullary regions or cornual-tubal junctions of any animal. However, sperm were observed in 93 per cent of the proximal sections and 30 per cent of the middle sections; the percentage decrease was marked. In a series of matings involving younger and older females, observations were made of relative contraction rates of the reproductive tract musculature. Contractions were more evident in the younger than in the older females. Peristaltic and antiperistaltic movements were very prominent, the latter being more pronounced. With the naked eye one could see large masses of semen moved anteriorly by vigorous antiperistaltic actions. The mass appeared whitish in contrast to the pinkish character of the uterine musculature. Black and Asde1l 3 have reported that, in the rabbit, the semen is rapidly squirted through the small lumen. Cornual contractions were less evident during anestrus than during estrus. Artificially inseminated females in estrus showed results similar to those noted during normal breeding. Marked peristaltic contractions could be elicited by simply touching the cornual wall with an instrument. These induced contractions were more extensive than those occurring naturally.
DISCUSSION Sperm transport in the golden hamster is similar to that of other eutherian mammals, and the ascent in the female reproductive tract is remarkably rapid. The rapidity of sperm transport in the cow was demonstrated by VanDemark and Moeller7 and VanDemark and Hays.s Factors which contribute to the rapid transit of the male gametes within the female tract include the marked antiperistaltic movements of the uterine cornua, which are possibly aided by oxytocin. The nonselective action of the female tract results in the transport of both dead and live sperm at a similar rate.
YAMANAKA & SODERWALL
474
Fertility & Sterility
The presence of peristaltic and antiperistaltic activity of the cornua during heat was reported as early as 1845 by Bischoff.2 These contractile movements were not credited with contributing to sperm transport. However, in the hamster these contractions do exert a direct influence on the rate of sperm ascent. Large masses of spermatozoa are forced anteriorly by antiperistaltic actions. Evans 4 stated that after spermatozoa reach the uterotubal junction in the dog, they are moved through the oviducts without the aid of an antiperistaltic wave. However, in the rat, contraction waves may spread up the ducts even to the ovarian ends (Blandau and Money3). In the hamster, as in the dog, the antiperistaltic waves stopped at the cornual-tubal junction. The number of spermatozoa entering the oviducts in the hamster is exceedingly small compared to the number present in the cornua. Similar results were observed by Blandau and Money3 in the rat.
SUMMARY 1. Eight-five female golden hamsters were mated and sacrificed at intervals averaging approximately 2 minutes after ejaculation, and the number of spermatozoa present in various segments of the reproductive tract was determined. 2. Comparable results were obtained in the left and right uterine cornua. 3. The shortest time after mating at which spermatozoa were observed in the ampulla was 91 seconds. 4. Antiperistaltic cornual movements were found to be pronounced and effective in forcing the anterior passage of the sperm masses. Q. Spermatozoan transport was most rapid when young females were mated with young males and only slightly less rapid when senescent males were used. 6. When old females were mated, spermatozoa from old males were transported at a significantly lower rate than spermatozoa from young males. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Problemy Zhivotnovod. 2:73, 1938. Die Enttoicklungsgeschichte des Hunde-Eis. Sohn schweig, Friedrick Vieweg u, 1845. BLACK, D. L., and ASDELL, S. A. Am. J. Physiol. 192:63, 1958. BLANDAU, R. J., and MONEY, W. L. Anat. Rec. 90:255, 1944. EVANS, E. I. Am.]. Physiol. 105:287, 1933. SODERWALL, A. L. Proc. Oregon Acad. Sc. 1 :45, 1945. VANDEMARK, N. L., and MOELLER, A. N. Am.]. Physiol. 165:674, 1951. VANDEMARK, N. L., and HAYS, R. L. Fertil. & Steril. 5:131, 1954.
BESCHLEVNOV, A. V. BISCHOFF, T. L. W.
Brann-
t
THE
on the rate of sperm transport through the female reproductive tracts in different species of mammals reveals considerable variation in findings. With improvement of technics for such studies, more recent reports have shown that less time is required for spermatozoa to traverse the female reproductive tract than was previously thought. Beschlevnov1 found that spermatozoa in the cow enter the uterus in less than 40 minutes after ejaculation and reach the ovarian end of the uterus in 4 hours. On the other hand, VanDemark and Moeller7 observed spermatozoa at the ovarian end of the fallopian tube in the cow in less than 3 minutes after artificial insemination to the midcervical region. In studies with the albino rat, Blandau and Monet noted spermatozoa in the uterine segments of the oviducts within 15 minutes after ejaculation in 21 per cent of the females; in one hour sperm were observed throughout the oviducts of all the mated rats. The work by VanDemark and Moeller7 indicates that the rate of spermatozoan transport is much greater than would be expected if sperm motility were the only factor involved in their movement within the female reproductive tract. The investigation reported here was carried out to determine the time required for spermatozoa to reach various levels of the female genital tract in the hamster (Mesocricetus auratus), and the mechanism of their transport. LITERATURE
From the Department of Biology, University of Oregon, Eugene, Ore. This study was aided by grants from the U. S. Public Health Services (No. 4213), and Faculty Research Committee of the University of Oregon. 470
Vol. 11, No.5, 1960
SPERM TRANSPORT
471
MATERIALS AND METHODS The data in this study were obtained from matings involving 85 female hamsters ranging in age from 2 to 21 months and 13 male hamsters of similar age range, used for matings. The animals were divided into 2 age groups: the young, consisting of animals ranging in age from 2 to 12 months, and the old, from 13 to 21 months. From these, 4 breeding groups were established (Table 1). Females in heat were placed in breeding boxes with vigorous males, as described by SoderwalL 6 The male hamster will ordinarily mount a female in heat a number of times before ejaculation. In order to observe the exact time of ejaculation, the male was allowed to mount a teaser female 10 times. During this period an experimental female, in heat, was placed in an adjacent box and after the tenth mounting the teased male was placed with the female. The exact moment of ejaculation was timed with a stop watch. The mated female was killed by a blow on the back of the head at intervals ranging from 1 to 2~~ minutes following ejaculation. The abdomen was opened immediately and the genital tract exposed. Thin hemostats were placed at the cornual-cervical junction (proximal third), midcornual (mid third), distal-cornual (distal third), and ampullary portion of the uterine tube (Fig. 1). The total elapsed time for this procedure was approximately 15 seconds. The cornual segments were isolated and the contents of the lumen were squeezed onto a clean glass slide and diluted 1: 200 parts with Locke's
Section Third
Fig. 1. Diagrammatic view of female hamster reproductive tract. Dashes indicate location of hemostats.
472
Fertility & Sterility
YAMANAKA & SODERWALL
solution in a blood pipet; counting was done with a hemocytometer. The ampullae were rinsed thoroughly in Locke's solution and observed for the presence of spermatozoa. OBSERVATIONS Table 1 summarizes the essential data and shows the distribution of spermatozoa within the reproductive tract in females killed on an average of approximately 2 minutes after ejaculation. TABLE 1.
Sperm Transport Times in Female Reproductive Tract Cornual segments with sperm
Group
Average No. age females (months) used
Proximal L. R. %
Middle L.
R.
%
L.
Distal
R. %
Average Average Ampulla time time (sec.).' L. R. % (sec.)t
1 Young female
8
X Young male
27
27 27 100 24 25 90 16 15 57
105
8 7 26
115
20
20 19
6 35
120
3 1 10
155
18
18 18 100 15 15 83
9 11 55
115
5 5 22
127
20
19 18
0
120
o
9
2 Old female
15
X Young male
98
12 13 62.3 8
10
3 Young female
9
X Old male
15
4 Old female
18
X Old male
92.5 7
5 30
0
0
0
0
17
• Average time of sacrifice for whole grouP.
t Average time of sacrifice tor animals in which sperm were found in the ampulla.
Group 1.
In matings between males and females of 8 to 9 months of age, all of the 27 animals sacrificed showed spermatozoa in all parts of the cornua at the end of 2 minutes. Fifty-four cornua were examined at an average of 105 seconds; 100 per cent showed spermatozoa at the proximal end, 90 per cent at the middle portion, and 67 per cent at the distal end. Twenty-six per cent of the 54 ovarian segments of the cornua contained 1-200 spermatozoa. The average time of examination in these cases was 115 seconds. However, in one instance, 4 spermatozoa were observed in this region after H minutes. Group 2. Twenty old females were mated with young males and killed at an average time of 120 seconds after ejaculation. Spermatozoa were present in 98, 62, and 35 per cent in the proximal, middle, and distal sections
Vol. II, No.5, 1960
SPERM TlIANSPORT
473
of the uterine cornua, respectively. In the ampullary region, 10 per cent of the sections were infiltrated by spermatozoa; the average time in these instances was 155 seconds. Group 3. In the 18 pairs of cornua from young females bred with old males, the results were much like those in Group 1. At the average sacrifice time of 115 seconds, 100, 83, and 55 per cent showed spermatozoa at the proximal, mid, and distal sections, respectively. In the ampullary region, 22 per cent showed positive results at an average of 127 seconds. Group 4. Twenty old females were mated with old males and the females sacrificed at an average of 2 minutes after ejaculation. Spermatozoa were not noted in the ampullary regions or cornual-tubal junctions of any animal. However, sperm were observed in 93 per cent of the proximal sections and 30 per cent of the middle sections; the percentage decrease was marked. In a series of matings involving younger and older females, observations were made of relative contraction rates of the reproductive tract musculature. Contractions were more evident in the younger than in the older females. Peristaltic and antiperistaltic movements were very prominent, the latter being more pronounced. With the naked eye one could see large masses of semen moved anteriorly by vigorous antiperistaltic actions. The mass appeared whitish in contrast to the pinkish character of the uterine musculature. Black and Asde1l 3 have reported that, in the rabbit, the semen is rapidly squirted through the small lumen. Cornual contractions were less evident during anestrus than during estrus. Artificially inseminated females in estrus showed results similar to those noted during normal breeding. Marked peristaltic contractions could be elicited by simply touching the cornual wall with an instrument. These induced contractions were more extensive than those occurring naturally.
DISCUSSION Sperm transport in the golden hamster is similar to that of other eutherian mammals, and the ascent in the female reproductive tract is remarkably rapid. The rapidity of sperm transport in the cow was demonstrated by VanDemark and Moeller7 and VanDemark and Hays.s Factors which contribute to the rapid transit of the male gametes within the female tract include the marked antiperistaltic movements of the uterine cornua, which are possibly aided by oxytocin. The nonselective action of the female tract results in the transport of both dead and live sperm at a similar rate.
YAMANAKA & SODERWALL
474
Fertility & Sterility
The presence of peristaltic and antiperistaltic activity of the cornua during heat was reported as early as 1845 by Bischoff.2 These contractile movements were not credited with contributing to sperm transport. However, in the hamster these contractions do exert a direct influence on the rate of sperm ascent. Large masses of spermatozoa are forced anteriorly by antiperistaltic actions. Evans 4 stated that after spermatozoa reach the uterotubal junction in the dog, they are moved through the oviducts without the aid of an antiperistaltic wave. However, in the rat, contraction waves may spread up the ducts even to the ovarian ends (Blandau and Money3). In the hamster, as in the dog, the antiperistaltic waves stopped at the cornual-tubal junction. The number of spermatozoa entering the oviducts in the hamster is exceedingly small compared to the number present in the cornua. Similar results were observed by Blandau and Money3 in the rat.
SUMMARY 1. Eight-five female golden hamsters were mated and sacrificed at intervals averaging approximately 2 minutes after ejaculation, and the number of spermatozoa present in various segments of the reproductive tract was determined. 2. Comparable results were obtained in the left and right uterine cornua. 3. The shortest time after mating at which spermatozoa were observed in the ampulla was 91 seconds. 4. Antiperistaltic cornual movements were found to be pronounced and effective in forcing the anterior passage of the sperm masses. Q. Spermatozoan transport was most rapid when young females were mated with young males and only slightly less rapid when senescent males were used. 6. When old females were mated, spermatozoa from old males were transported at a significantly lower rate than spermatozoa from young males. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Problemy Zhivotnovod. 2:73, 1938. Die Enttoicklungsgeschichte des Hunde-Eis. Sohn schweig, Friedrick Vieweg u, 1845. BLACK, D. L., and ASDELL, S. A. Am. J. Physiol. 192:63, 1958. BLANDAU, R. J., and MONEY, W. L. Anat. Rec. 90:255, 1944. EVANS, E. I. Am.]. Physiol. 105:287, 1933. SODERWALL, A. L. Proc. Oregon Acad. Sc. 1 :45, 1945. VANDEMARK, N. L., and MOELLER, A. N. Am.]. Physiol. 165:674, 1951. VANDEMARK, N. L., and HAYS, R. L. Fertil. & Steril. 5:131, 1954.
BESCHLEVNOV, A. V. BISCHOFF, T. L. W.
Brann-
t