SPERM TRANSPORT AND DISTRIBUTION IN THE REPRODUCTIVE TRACT OF THE FEMALE RABBIT AFTER INTRAPERITONEAL INSEMINATION*

Transcription

1 FERTLTY AND STERLTY Copyright 1974 The American Fertility Society Vol. 25, No. 12, December 1974 Printed in U.s.A. SPERM TRANSPORT AND DSTRBUTON N THE REPRODUCTVE TRACT OF THE FEMALE RABBT AFTER NTRAPERTONEAL NSEMNATON* G. C. VANDEN BOSCH, M.S., AND E. S. E. HAFEZ, PH.D. (Cantab.) Departments of Gynecology-Obstetrics and Physiology, Wayne State University School of Medicine, Detroit, Michigan 4821 Although intraperitoneal insemination has been used extensively and successfully in birds,l very little research has been done using the techniques with mammals. Most research with mammalian intraperitoneal insemination has focused on the observation of fertilization. Hadek2 performed the first intraperitoneal insemination in rabbits. The conception rate was high only if the insemination was performed eight hours before ovulation and if the spermatozoa were thoroughly washed. Mroueh and Mastroianni,3 however, did recover spermatozoa from the rabbit oviduct and uterus, but they did not evaluate parameters such as the number of spermatozoa inseminated or the exact location of insemination. Adams,4 after varying the number of spermatozoa inseminated, postulated that the large number of spermatozoa in the rabbit oviduct caused polyspermy and subsequent reduction of implantation. Research with the rhesus monkey after intraperitoneal insemination, however, indicates that larger numbers of spermatozoa ensure better fertilization.; Also, since 1% of all human infertility is due to cervical factors after vaginal insemination 6 and since spermatozoa have been found in the peritoneal cavity after vaginal insemination/,h further research is warranted. Received October 22, * Supported by The Ford Foundation grant no and the National nstitute of Child Health and Human Development grant no. HD6234. The purpose of this study was to define the distribution of spermatozoa within the oviduct after intraperitoneal insemination and to correlate it to such factors as sperm motility and oviductal contractility. Penetration of eggs by spermatozoa and fertilization were not studied since these parameters were adequately examined by other investigators.2'4 MATERALS AND METHODS Twenty mature female New Zealand rabbits were divided into three groups. The six rabbits in group 1 were in estrus and were not treated. They were considered to be in estrus if the vulva was purple. The eight rabbits in group 2 received 25 V of human chorionic gonadotropin (HCG) 12 hours prior to insemination. The eight rabbits in group 3 received 25 V of HCG 96 hours prior to insemination. Semen was collected from three healthy bucks with the use of an artificial vagina and treated in one of two ways: For insemination with live spermatozoa, the semen was analyzed for sperm motility and density. fat least 8% of the spermatozoa were motile in a forward direction, two aliquots of the semen, each containing 1 x 1 8 spermatozoa, were withdrawn with 1 ml syringes. For insemination with dead spermatozoa, the semen was gently centrifuged for ten minutes to separate the spermatozoa and seminal plasma. The supernatant (seminal plasma) was drawn off and the sperm were mixed with 139

2 14 VANDEN BOSCH AND HAFEZ December 1974 UTERUS UTJ \._ ") V OVDUCT FG. 1. Segments into which the reproductive tract was divided before flushing. UTJ = uterotubal junction..1 M solution of copper sulfate for ten minutes. The solution was again gently centrifuged for ten minutes to separate the spermatozoa and copper sulfate. The copper sulfate was withdrawn and the seminal plasma was mixed with the spermatozoa. The semen was then analyzed to assure that there was no motility and to determine the density of the spermatozoa in the semen. Two aliquots, each containing 1 x 1 8 spermatozoa, were withdrawn with 1 ml syringes. ntraperitoneal insemination was accomplished by laparotomy through a midline incision 7 cm long. The rabbit was anesthetized with an intravenous injection of sodium pentobarbital (Veterinary Laboratories) in the marginal vein of the ear. When the ovary was visible, the aliquot of semen containing 1 x 1 8 spermatozoa was slowly deposited dorsal to the ovary, but not into the fimbriae of the oviduct. This same procedure was performed on the contralateral side, after which the abdominal wall was closed with continuous chromic suture, and the skin was closed with wound clips. Half of the does of each group were inseminated with at least 8% motile spermatozoa and the other half with nonmotile spermatozoa. Five hours after insemination, the does were killed with an intravenous overdose of sodium pentobarbital. The whole reproductive tract was removed and trimmed of fat and connective tissue. The oviduct was then measured and divided into four equal segments, the first segment beginning with the fimbriated end and the fourth segment terminating at the uterotubal junction. The uterotubal junction and uterus were separated; thus, the entire reproductive tract was divided into oviductal segments, both uterotubal junctions, and the cervices and vagina (Fig. 1). A 2.5 ml disposable syringe and a 24 gauge needle were used to flush each portion of the tract with 2. ml of.9/ NaC solution and.5 ml of air into graduated 15 ml centrifuge tubes. 9 Between each flushing the needle was thoroughly cleaned to minimize any contamination from one segment to the next. The flushings were centrifuged at 1,5 g for ten minutes and 1.6 ml of the supernatant was removed immediately after centrifugation. Then,.1 ml of 5./ eosin solution was added to the remaining sperm concentrate; thus, the final volume was.5 ml. Each volume was thoroughly mixed with a clean glass pipette and the number of spermatozoa in each segment was determined with a Fuchs-Rosenthal Ultraplane hemocytometer. One centimeter segments of three oviducts and one uterus, having been flushed previously, and eight fimbriae were fixed

3 Vol. 25, No. 12 SPERM TRANSPORT AND DSTRBUTON 141 N a.. U) - => z NSEMNATON WTH DEAD SPERMATOZA o NSEMNATON WTH LVE SPERMATOZA r6 : 2 3 4: UTERO- UTERUS TUBAL -OVDUCT-JUNCTON, SEGMENT FG. 2. Average number of spermatozoa recovered from the various segments of the estrous reproductive tract following intraperitoneal insemination. N a.. U) 8-6 => z 4 1 "" o NSEMNATON WTH DEAO SPERMATOZOA NSEMNATON W TH LVE SPERMATOZ OA in 1% buffered formalin phosphate for two days and embedded in paraffin blocks. Longitudinal serial sections were obtained and stained with Harris's hematoxylin and eosin. The sections were examined microscopically for spermatozoa and their orientations within the oviducts. RESULTS The ovulation rate in the HeG-treated rabbits varied from 4 to 16 with an average of 8.. The length of the oviducts ranged from 9. to 18.8 cm with an average of 13.2 cm. The length of the dissected uterine horn ranged from 6. to 15. cm with an average of 8.9 cm. No correlation could be found between the length of the oviduct or uterus and the number of spermatozoa recovered from these organs. Recovery of spermatozoa after intraperitoneal insemination. ntraperitoneal insemination was performed during three N a.. 1 U) 'l; SOD => z 6 4 Q NSEMNATON WTH DEAD SPERMATOZOA o NSEMNATON WTH LVE SPERMATOZA X NO SPERMATOZA RECOVERED 2 1m : 2 3 4: UTERO- UTERUS :_ OVDUCT-: TUBAL, JUNCTON SEGMENT FG. 3. Average number of spermatozoa recovered from the various segments of the female reproductive tract following intraperitoneal insemination 12 hours after HCG injection. 2 x : 2 3 4: UTERO- UTERUS :-OVDUCT-' TUBAL : JUNCTON SEGMENT FG. 4. Average number of spermatozoa recovered from the various segments of the female reproductive tract following intraperitoneal insemination 96 hours after HCG injection.

4 142 VANDEN BOSCH AND HAFEZ December 1974 HORMONAL STATUS TYPE of SPERMATOZOA NSEMNATED ESTRUS { LVE FH1J111HD11114 DEAD OVULATNG { PSEUOOPREGNANT { F'Mffff& "WM 1ij L- llllllwlll11 LVE DEAD LVE DEAD o PERCENTAGE TD UTERUS. OVDUCT.1 a UTERO- TUBAL JUNCTON FG. 5. Sperm distribution in the female reproductive tract at various hormonal stages and after intraperitoneal insemination with live or dead spermatozoa as a percentage of the total number of spermatozoa recovered by flushing. hormonal stages: estrus, pseudopregnancy, and ovulation. More spermatozoa were consistently recovered from the reproductive tract after insemination with live spermatozoa than with dead spermatozoa. During estrus and ovulation, more than twice the amount was recovered after insemination with live rather than dead spermatozoa; however, during pseudopregnancy slightly less than twice the amount was recovered after insemination with live rather than dead spermatozoa. n addition, more spermatozoa were recovered from each segment of the oviduct after insemination with live rather than dead spermatozoa (Figs. 2 to 4). The same relationship was evident in the uterotubal junction and uterus. However, the uterotubal junction of pseudopregnant rabbits contained no spermatozoa in either case. Considerably fewer spermatozoa were found in the reproductive tract of pseudopregnant rabbits than in that of rabbits at either estrus or ovulation. The average numbers of spermatozoa recovered at estrus and ovulation after insemination with live spermatozoa were 7,13 and 6,74, respectively, while the number recovered at pseudopregnancy was only 3,75 afterinsemination with live spermatozoa. Similar results were obtained after insemination with dead spermatozoa. At estrus and ovulation, 2,5 and 2,51 spermatozoa were recovered; however, at pseudopregnancy, only 1,93 spermatozoa were recovered. The possible presence of spermatozoa

5 Vol. 25, No. 12 SPERM TRANSPORT AND DSTRffiUTON 143 in both cervices and vagina was checked in 12 rabbits but was found in only one rabbit. The number from both organs was 16. Sperm distribution after intraperitoneal insemination. A gradient in the number of spermatozoa existed between the ampulla (segments 1 and 2) and the isthmus (segments 3 and 4). A larger number of spermatozoa resided in the ampulla in all cases. The largest percentage of spermatozoa resided in segment 1; this ranged from 38% after intraperitoneal insemination with dead spermatozoa during estrus and ovulation to 83% after insemination with dead spermatozoa during pseudopregnancy (Fig. 5). A gradient in the number of spermatozoa through all segments of the reproductive tract was evident during ovulation after insemination with live spermatozoa. The number and percentage of spermatozoa constantly declined from segment 1 (42%) to the uterotubal junction and uterus (8%). Such a gradient was not observed after insemination during estrus or pseudopregnancy. Although sperm motility appeared to affect the number of spermatozoa transported to the oviduct, it apparently did not affect their distribution within the reproductive tract, at least during estrus and ovulation. With a few exceptions, the percentage of spermatozoa in each segment was similar during both estrus and ovulation and after insemination with either live or dead spermatozoa. Pseudopregnancy, on the other hand, which caused a reduction in the number of spermatozoa in the reproductive tract, also caused the retention of a large number of spermatozoa in the ampulla. After insemination with dead spermatozoa during this period, 1% of the recovered spermatozoa was found in the ampulla after insemination with live spermatozoa. These are inordinately large percentages when compared to sperm distribution during either estrus or ovulation. Histology of the rabbit reproductive tract after intraperitoneal insemination. Histologic sections of flushed oviducts and uterus were examined for spermatozoa in order to test the effectiveness of flushing in removing spermatozoa. No spermatozoa could be found in segments of the oviduct or in the uterus, all of which contained spermatozoa before flushing. There was tearing and shredding of the mucosal epithelium, indicating that the force created within the oviduct by flushing was more than necessary for effective removal of spermatozoa. Serial sections of eight fimbriae were also examined to help determine the function of this structure on sperm transport and distribution after intraperitoneal insemination. Unfortunately, due either to the fixing or staining process, large numbers of spermatozoa were found in only two fimbriae. One fimbria, obtained from a pseudopregnant rabbit after insemination with dead spermatozoa, contained 598 spermatozoa. Spermatozoa were usually found in groups of five or more in both fimbriae. These groups of spermatozoa appeared to have no particular orientation and, thus, appeared trapped within the mucosal folds. DSCUSSON Parker,O discussing the mechanism of sperm transport in the oviduct, did mention that spermatozoa could be transported toward the uterus, but his major emphasis was on sperm transport toward the ovary. Since that time, discussions of sperm transport within the oviduct have concerned themselves primarily with unidirectional movement toward the ovary as opposed to ovum transport toward the uterus. However, from the present study and from other studies utilizing intraperitoneal insemination, it is evident that spermatozoa are, indeed, transported toward the uterus as well as

6 VANDEN BOSCH AND HAFEZ December 1974 toward the ovary. Thus, spermatozoa are transported within the oviduct with a dynamic force capable of either an adovarian or abovarian direction. The distribution of spermatozoa, however, does not completely reflect this dual ability of the oviduct. After vaginal insemination of the rabbit,9 mouse,ll pig,12 spermatozoa are concentrated within the uterotubal junction and isthmus. n the present study, most of the spermatozoa were recovered from the ampulla. t is conceivable that the isthmus is responsible for both of these events since studies have demonstrated that it acts as a barrier to sperm transport within the oviduct After vaginal insemination, passage of spermatozoa would be restricted from the ampulla to the isthmus. After intraperitoneal insemination, the fimbriae, also, may facilitate the retention of spermatozoa in the ampulla. Histologic examination of serial sections of the fimbriae demonstrated groups of spermatozoa with no particular orientation, apparently trapped within the mucosal folds. The isthmus and uterotubal junction, to allow transport of ova into the uterus, are inoperative as gamete barriers by the fourth day after ovulation and would be expected to reduce the difference in the number of spermatozoa between the ampulla and isthmus. However, after intraperitoneal insemination during the fourth day after ovulation, an increase in this difference was observed. This would suggest that some factor, other than the barriers created by the isthmus and uterotubal junction, affects the distribution of spermatozoa. f the anatomical barriers to sperm transport during estrus and ovulation are no longer functional, some qualitative and/or quantitative change in the oviduct must take place to create the difference in the number of spermatozoa between the ampulla and isthmus. Oviductal contractility is likely since the contractile pattern is modified during the luteal phase of the reproductive cycle. By the fourth day after ovulation in the rabbit, the number and intensity of contractions decrease During the days following ovulation, similar reductions in the oviductal contractions occur in women19 and rhesus monkeys.2 Also, by the third and fourth days of pseudopregnancy in the rabbit, the number of antiperistaltic contractions increases.21 Thus, it is possible that oviductal contractility under hormonal regulation has the ability to affect sperm distribution within the oviduct. t is probable that the large number of spermatozoa in the ampulla may lead to low implantation rates after fertilization. t has been postulated that a low implantation rate following intraperitoneal insemination may be due to increased polyspermy and subsequent degeneration of ova.4 n the present experiment, after insemination with live spermatozoa during ovulation, an average number of 4,3 spermatozoa was recovered from the ampulla; this is well above the number recovered (1,972) after vaginal insemination.9 Mroueh and MastroiannP also recovered an average 13, spermatozoa from the oviduct after intraperitoneal insemination in the rabbit. This is also much greater than the average number (2, to 6,) recovered after vaginal insemination in the rabbit.9.22 Thus, since the total number of spermatozoa in the oviduct and the number in the ampulla are far greater than the number of spermatozoa recovered after vaginal insemination, it is feasible that polyspermy may result from intraperitoneal insemination. Motile spermatozoa were more effective in gaining entrance into the oviduct than immotile spermatozoa, suggesting that spermatozoa are aided by their own motility. However, the mechanism of entry into the oviduct depends on other parameters since substantial amounts of immotile spermatozoa were recovered

7 Vol. 25, No. 12 SPERM TRANSPORT AND DlSTRmUTON 145 from the oviducts, and since inert particles such as starch have been transported through the reproductive tract after deposition in the peritoneal cavity.23 From the present study, further evidence of other factors in the entry mechanism arises from the observation that pseudopregnancy caused a reduction in the number of spermatozoa recovered from the oviduct. This suggests that there are cyclic changes in the ability of the oviduct to recover particles and gametes from the peritoneal cavity. njection of carbon particles in mouse24 and crayfish eggs and starch particles in the rabbit25 indicates that the pickup mechanism is most effective at about the time of ovulation but that it functions to some degree throughout the reproductive cycle. Pseudopregnancy appears to be the most ineffective period for particle pickup, possibly because of a change in contractile pattern of the oviduct. 26 Further research is needed to clarify several physiologic aspects of sperm transport: s polyspermy the only cause of the low implantation rate after intraperitoneal insemination? What is the number of spermatozoa needed for intraperitoneal insemination which would ensure fertilization but prevent polyspermy? What are the different factors affecting sperm distribution within the oviduct? How does the change in the balance of ovarian steroids affect sperm distribution? SUMMARY Mature female rabbits were divided into three gtoups: (1) untreated and in estrus, (2) injected with HCG 12 hours prior to insemination, and (3) injected with HCG 96 hours prior to insemination. Each group was inseminated intraperitoneally with either 2 x 1 8 live or dead spermatozoa. Five hours after insemination, the rabbits were autopsied and the spermatozoa were recovered by flushing the segmented reproductive tract. n addition, serial sections were obtained from several fimbriae and from several segments of the reproductive tract which had been flushed previously. t was evident that spermatowa were transported both in an abo varian and adovarian direction in the oviduct and that several factors affected sperm transport. More spermatozoa were consistently recovered from the reproductive tract after insemination with motile spermatozoa than with immotile spermatozoa. Also, the number of spermatozoa found in the reproductive tract of pseudopregnant rabbits was considerably less than the number recovered at either estrus or ovulation. Thus, it would appear that the pickup of spermatozoa by the fimbriae depends on both sperm motility and oviductal contractility. The majority of spermatozoa recovered from the oviduct were found in the ampulla. This difference between the number of spermatozoa in the ampulla and that in the isthmus can be explained on the basis of anatomical barriers in the oviduct created by the isthmus. However, since pseudopregnancy exaggerates the gradient in the number of spermatozoa, oviductal contractility is also implicated in the mechanism of sperm distribution within the oviduct. REFERENCES 1. Van Drimmelen GC: Artificial insemination of birds by the intraperitoneal route. Onderstepoort J Vet Res (Suppl 1): 1, Hadek R: ntraperitoneal insemination of rabbit does. Proc Soc Exp Bio Med 99:39, Mroueh A, Mastroianni L Jr: nsemination via the intraperitoneal route in rabbits. Fertil Steril 17:76, Adams CE: ntraperitoneal insemination in the rabbit. J Reprod Fertil 18:333, Van Pelt LF: ntraperitoneal insemination of Macaca Mulatta. Fertil Steril 21:159, Moghissi KS: Sperm migration through the human cervix. n Biology of the Cervix. Edited by RJ Blandau, KS Moghissi. Chicago, Chicago Press, 1973, p 35

8 146 VANDEN BOSCH AND HAFEZ December Horne HW Jr, Thibault J: Sperm migration thro.ugh the human female reproductive tract. Fertil Steril 13:444, Ahlgran M: Migration of spermatozoa to the fallopian tubes and the abdominal cavity in women including some immunological aspects. Studentlitterature. Lund, 1969, p Braden AWH: Distribution of sperm in the genital tract of the female rabbit after coitus. Aust J Bio Sci 6:693, Parker GH: The passage of the spermatozoa and ova through the oviducts of the rabbit. Proc Soc Exp Bio Med 27:826, Ods PJ: Effect of the T-Locus on sperm distribution in the house mouse. Bio Reprod 2: 91, Rigby JP: The distribution of spermatozoa in the genital tract of the sow. Ph.D. Thesis, Univ of Liverpool, Liverpool, Brundin J: Distribution and function of adrenergic nerves in the rabbit fallopian tube. Acta Physiol Scand 66:5, Hunter RHF, Leglise PC: Polyspermic fertilization following tubal pregnancy in pigs with particular reference to the role of the isthmus. J Reprod Fertil 24:233, Zamboni L: Fertilization in the mouse. n Biology of Mammalian Fertilization and mplantation. Edited by KS Moghissi, ESE Hafez. Springfield ll, CC Thomas, 1972, p Harper MJK: Stimulation of sperm movement from the isthmus to the site of fertilization in the rabbit oviduct. Bio Reprod 8:369, Salomy M,Harper MJK: Cyclical changes of oviduct motility in rabbits. Bio Reprod 4:185, Aref, Hafez ESE: Cyclical changes in uterooviductal motility in the rabbit. J Reprod Fertil 32:93, Rubin C: The influence of hormonal activity of the ovaries upon the character of tubal contractions as determined by uterine insufflation. Am J Obstet Gynecol 37:394, Seckinger DL, Corner GW: Cyclic variations in the spontaneous contractions of the human fallopian tube. Bull Johns Hopkins Hosp 39: 371, Westman A: A contribution to the question of the transit of the ovum from the ovary to uterus in rabbits. Acta Obstet Gynecol Scand 5 (Suppl 3):1, Chang MC: Fertilization in relation to the number of spermatozoa in the fallopian tubes of rabbits. Annal Obstet Gynecol 73:918, Decker A, Decker WH: A tubal function test. Obstet Gynecol 4:35, Wimsatt WA, Waldo CM: The normal occurrence of a peritoneal opening in the bursa ovarii of the mouse. Anat Rec 93:47, Anderes E: Zur frage des eintransportes durch die tube. Schwiez Med Wochenschr 71:364, Chang MC: Fertilization, transportation, and degeneration of eggs in the pseudopregnant or progesterone-treated rabbit. Endocrinology 84:356, 1969