I nfluence of Semen on the Motility of the Uterus in the Guinea Pig

Transcription

1 I nfluence of Semen on the Motility of the Uterus in the Guinea Pig In-Vitro Studies M. FREUND, PH.D., AND ALBERT M. LEFKOVITS, A.B. AN UNANSWERED QUESTION lo on the physiology of reproduction is: "Are there substances in the seminal fluid that affect the motility of the female reproductive tract in species other than man?" As a result of concurrent studies 5 6 on semen production in guinea pigs, we had available a regular supply of liquefied guinea pig semen adequate for an investigation that might answer this question. Kurzrok and Lieb, and Cockrill et al. reported both increase and decrease in the motility of human uterine strips after the addition of human seminal plasma and suggested that the differences in response might be due to the varying properties of seminal plasma in different men. Goldblatt found that human seminal plasma had a markedly stimulating effect on the contractions of the guinea pig uterus in vitro. Euler 4 isolated a substance from human seminal plasma which he called prostaglandin and showed that it stimulated the motility of the isolated uterus or uterine strips from cow, rabbit, guinea pig, rat, and human. He reported 3 that prostaglandin could be isolated from the semen of men and of rams but not from bulls, boars, dogs, rabbits, or guinea pigs. More recently, Karlson used internal tocometry to demonstrate that human semen deposited in the vagina caused contraction of the nonpregnant human uterus. Using similar recording technics, Eliasson and Posse reported that human prostaglandin exerts the same effects on the contractions of the human uterus as does the whole semen. Since prostaglandin has been isolated only from the seminal plasma of man and of sheep, the question of whether the semen of other animals has a stimulating effect on the uterine motility of females of the same species 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 Grant RG-6014 and, in part, by Grant RG-6880 from the Division of General Medical Sciences, U. S. Public Health Service. 459

2 460 FREUND & LEFKOVITS FERTILITY & STERILITY remains unanswered. The availability of guinea pig semen and uteri made possible the choice of this species for these studies. MATERIALS AND METHODS Young virgin female guinea pigs of the English breed (Rockland Farms), 3-4 weeks old, were housed on wire grilles and maintained on Purina guinea pig pellets, greens daily, and tap water. Mter the animals had reached sexual maturity (400 gm.), they were examined each day for evidence of estrus, i.e., for the opening of the vaginal skin and for the presence of vaginal mucus. The mean body weight of the guinea pigs used in this study was 481 gm. and the mean uterine weight (including small segments of the uterine horns, the cervix, and small segments of the vagina) was 860 mg. The guinea pig semen was collected by electro ejaculation into 1 ml. of phosphate buffer containing 1 mg. chymotrypsin, as previously described. 5 The chymotrypsin phosphate prevented formation of the semen coagulum ("rodent vaginal plug") produced after ejaculation. Preliminary work indicated that the chymotrypsin phosphate, by itself, had no effect on uterine motility and also that semen in chymotrypsin phosphate had the same effect as an equivalent amount of raw liquid semen. When liquid semen was ejaculated, it was used directly; but, in general, the semen coagulated after ejaculation unless collected in the chymotrypsin phosphate. Each female was sacrificed by a blow on the head and the uterus was dissected free. One thread was passed through the vagina just below the cervix and another through both of the uterine horns just above the point where they leave the body of the uterus. Both threads were tied as loops so that the lumina were not occluded. Every attempt was made to avoid handling or stretching the body of the uterus. The preparation was cut just above and below the ties and rapidly mounted in a 50-ml. constant-temperature bath (39 ± 1) of Locke's solution. The Locke's solution was replaced at the rate of 5 ml./min., and a gas mixture of 95% oxygen and 5% carbon dioxide was continuously bubbled through the bath. Recordings of the contractions were made on a long paper kymograph, using a light muscle lever with a 3:1 magnification. The pattern of spontaneous regular conh"action, characteristic of the estrus guinea pig uterus in vitro, began within min. and a 30-min. control record was then obtained. After this control period was completed, the fluid level in the bath was lowered without exposing the uterus to the air, and 0.4 ml. of raw semen or 1.0 ml. of the semen-chymotrypsin phosphate mixture was added. The immediate and long-term responses of the uterus were recorded and, as a rule, the contractions were followed for 1-2 hours after the semen was added.

3 VOL. 12, No.5, 1961 SEMEN AND MOTILITY 461 The volume of semen ejaculated by the guinea pig during coitus is unknown. Therefore, the mean volume of semen collected by electroejaculation (0.4 m!.) was used as the standard dose of semen in this study. Varying doses of semen were added to 4 uteri in a preliminary effort to determine the dose-response relationship. In each case, the addition of smaller volumes of semen (0.12 and 0.18 m!.) resulted in the same type and magnitude of response by the uteri, as was the case when the standard dose (0.4 m!.) was added. There is no evidence to suggest the existence of a dose-response relationship within the dosage range studied. A 30-min. control period and a 30-min. period, after semen was added, were selected and the amplitude, frequency, and duration of contraction were measured and compared. Mean amplitude was determined from measurements of each individual contraction during a period. Frequency was expressed as the number of discrete contractions per 30-min. period. Where it was not possible to measure a complete 30-min. period, shorter periods were used and values were expressed in terms of a 30-min. period. When the contractions were regular and continuous, mean duration was calculated as the reciprocal of the frequency; otherwise, measurement of individual contractions was made and converted to minutes by use of the time record on the kymograph paper. RESULTS Preliminary work indicated that a spontaneous pattern of regular contractions in vitro was characteristic of the uteri of guinea pigs in estrus (+ 1 day), while uteri from those in other stages of the estrous cycle gave little or no evidence of spontaneous motility. The nonestrus uterus exhibited little motility, gave little or no response to histamine or acetylcholine, and did not respond to the addition of guinea pig semen. The data from 15 trials of the addition of semen to the estrus uterus of the guinea pig demonstrate that the semen causes a marked increase in the motility of the uterus. (Table 1). After the addition of the semen, there was an immediate and characteristic response, consisting of sustained contractions of high tone which lasted approximately 5-10 min. After this short initial period of response, contractions returned to the baseline level and a new pattern was established (cf. Fig. 1). This new pattern was characterized by a marked increase in the amplitude of contraction (+91 per cent), a decrease in the frequency of contraction (-28 per cent), and a marked increase in duration of contraction (+55 per cent). The response of the estrus uterus was very consistent (Table 1) and the new pattern of contractions persisted for long periods (as long as 3 hours in

4 162 FREUND & LEFKOVITS FERTILITY & STERILITY TABLE 1. Mean amplitude (mm.) Post- Record Control addition % no. period period change MEAN % INCREASE: 91 * Contractions per 30 min. Contractile Response of Estrus Guinea Pig Uterus to Semen Frequency * Post- Control addition % period period change MEAN % DECREASE: 28 Mean duration (min.) Post- Control addition % period period change MEAN % INCREASE: 55 I o 10 min. 20 min. I 30 min. I 40 min ml. semen added. 50 min. Fig. 1. Sample kymograph (Record No.2, Table 1) showing control period (a 30-min. period preliminary to semen addition) and response to 0.4 ml. raw semen by an estrus guinea pig uterus in vitro. Changes shown are characteristic of the responses of the 15 uteri reported in this study.

5 VOL. 12, No.5, 1961 SEMEN AND MOTILITY 463 some of the trials) despite the continuous perfusion with Locke's solution at a rate which replaced the total bath volume every 10 min. Each uterus served as its own control and measurements were made before and after the addition of semen. When no treatment was made, the estrus uterus exhibited a regular frequency of contraction of a constaut amplitude and a constant baseline for 2-3 hours. DISCUSSION This study demonstrates the marked effect of guinea pig semen on the motility of the estrus guinea pig uterus and suggests the existence of several physiological relationships. The initial period of sustained contractions of high tone, immediately after the addition of the semen, may playa role in the movement of the sperm in the female reproductive tract of the guinea pig after coitus and is consistent with the knowledge acquired from studies with humans and other species that sperm are found shortly after coitus throughout the female reproductive tract. The pharmacodynamic effect of semen, in increasing the amplitude and duration of contraction and in decreasing the frequency of contraction, lasted for as long as 2-3 hours, although there was continuous perfusion, which changed the bath 6 times an hour. Moreover, in 3 trials where the bath was rapidly and repeatedly washed out with Locke's solution, the uteri maintained their postaddition pattern of higher and slower contractions. This long-term effect would seem to suggest a change in the status of the myometrium rather than just a short-term, readily reversible effect. Semen has been shown to have a marked influence on uterine contractions both in vitro and in vivo in the humans,g aud this has been confirmed in vitro in the guinea pig in this study. Therefore, the relationship between the effect on uterine motility of semen and of oxytocin released under the stimulus of coitus remains to be elucidated. Certainly, the previous studies of the influence of oxytocin on the movement of sperm in the female reproductive tract, which have not accounted for the semen effect, must be reconsidered, e.g., in the cow, where it was reportedll that sperm were found in the upper part of the female reproductive tract 2--4 min. after artificial insemination or natural mating and where this was ascribed solely to the effects of oxytocin release. The limitations of the classic lever-and-kymograph method of recording uterine contractions have become evident during the course of this study. A displacement transducer-strain gauge amplifier-recorder system has been set up and the studies are being continued with this newer technic. These

6 464 FREUND & LEFKOVITS FERTILITY & STERILITY studies have been started on in vivo, preparations, using anesthetized laparotimized guinea pigs. The effects of semen on uterine contractions during various phases of the estrous cycle are under investigation. SUMMARY The addition of guinea pig semen to the estrus guinea pig uterus caused a marked increase in the amplitude and duration of uterine contraction and a declease in the frequency of contraction. There was an initial 5- to lo-min. period of sustained contraction of very high tone after the addition of semen. This was followed by a much longer period, lasting for as long as 2-3 hours, of contractions of high amplitude and long duration. These results indicate that there is a pharmacodynamic effect of semen on uterine motility in the guinea pig, that may playa role in sperm transport in the female reproductive tract. REFERENCES 1. COCKRILL, J. R., MILLER, E. G., JR., and KURZROCK, R. The substance in human seminal fluid affecting uterine muscle. Am.]. Physiol. 112:577, ELIASSON, R., and POSSE, N. The effect of prostiglandin on the non-pregnant human uterus. Acta obst. et gynec. scandinav. 39:112, EULER, U. S. VON Zur Kenntnis der pharmakologischen Wirkungen von Nativserkreten und Extrakten mannlicher accessorischer Geschlechtsdrusen. Arch. exper. Path. u. Pharmakol. 175:78, EULER, U. S. VON On the specific vaso-dilating and plain muscle stimulating substances from accessory genital glands in man and certain animals (prostaglandin and vesiglandin). ]. Physiol. 88:213, FREUND, M. Collection and liquefaction of guinea pig semen. Proc. Soc. Exper. Biol. & Med. 98:538, FREUND, M. Initiation and development of sem~n production in the guinea pig. In press. 7. GOLDBLATT, M. W. Properties of human seminal plasma. J. Physiol. 84:208, KARLsoN, S. The influence of seminal fluid on the motility of the non-pregnant human uterus. Acta obst. et gynec. scandinav. 38:503, KURZROK, R., and LIEB, C. Biochemical studies of human semen. II. The action of semen on the human uterus. Proc. Soc. Exper. Biol. & Med. 28:268, PLANNED PARENTHOOD FEDERATION Inventory of unanswered questions in physiological mechanisms concerned with conception. New York, VANDEMARK, N. L., and HAYS, R. L. Rapid sperm transport in the cow. Fertil. & Steril. 5:131,1954.