This dissertation has been microfilmed exactly as received 70-6816
KOONER, Dilbagh Singh, 1939- SOME FACTORS ASSOCIATED WITH THE RELEASE OF HYALURONIDASE FROM BOVINE AND RABBIT SPERMATOZOA UNDER CONDITIONS OF SIMULATED CAPACITATION.
The Ohio State University, Ph.D., 1969 Physiology
University Microfilms, Inc., Ann Arbor, Michigan SOME FACTORS ASSOCIATED WITH THE RELEASE OF HYALURONIDASE
FROM BOVINE AND RABBIT SPERMATOZOA UNDER CONDITIONS
OF SIMULATED CAPACITATION
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
Dilbagh Singh Kooner5 B.V.Sc. & A,H., M.Sc.Dairying
********
The Ohio State University 1969
Approved by
Department of Dairy Science ACKNOWLEDGMENTS
My sincere thanks are due to my adviser, Dr. T. M. Ludwick for his guidance during the course of this investigation and in writing this manuscript. I am also indebted to him for the interest, he took in making my two years of stay in U.S.A., a memorable and very enjoy able experience of my life.
My thanks are due to Dr. W. R. Gomes, Dr. H. C. Hines, and
Dr. W. J. Tyznik for permission to use their laboratory facilities.
I am indebted to Dr. Eldon Brum, Mr. William Crist, Mr. David
Davis, Mr. Donald Long, Mr. Earl Rader, and Mr. Mehar Singh who assisted me in some of the surgical techniques involved, in collection of semen, and for their valuable counsel on several matters, some of them directly related to this investigation. I am also thankful to
Mr. Charles Gaskin for help in computer programming for carrying out statistical analyses.
But for the financial support, I received from the Agency for
International Development, Department of State, U.S.A., and the Punjab
Agricultural University, Ludhiana, Punjab, India, this study would not have been possible. In this regard, I wish to especially thank
Dr. D. Sunderasan, Dean, Post-Graduate Studies, Punjab Agricultural
University, Ludhiana (India) and Dr. Mervin G. Smith, Assistant Dean and Coordinator, International Affairs, College of Agriculture and
Home Economics, The Ohio State University, Columbus. I am indebted to my dear mother who inspired and encouraged me to seek a much needed education in a foreign land, very far away from home.
Last but not the least, I am thankful to Mrs. Esther Crabtree for painstakingly typing this manuscript, and my wife, Diane who corrected this manuscript and gave some valuable suggestions.
iii VITA
November 10, 1939 . . . Born in village Jandusingha, District Jullundur, Punjab State, India
1960...... B.V.Sc. & A.H. (Bachelor of Veterinary Science and Animal Husbandry) from Vikram University, Ujjain, India
1960-1961 ...... Demonstrator of Veterinary Medicine, M.P. College of Veterinary Science and Animal Husbandry, Mhow, India
1961-1963 ...... Junior Fellow, Indian Council of Agricul tural Research, New Delhi, India
1963...... M.Sc.Dairying from Panjab University, Chandigarh, India
1963 . Demonstrator of Dairy Husbandry, College of Dairy Science, Karnal, India
1963-1964 ...... Veterinary Service Representative, Pfizer Private Limited, Bombay, India
1964...... Assistant Professor, Department of Animal Science, Punjab Agricultural University, Ludhiana, India
1967-1969 ...... Attended The Ohio State University, Columbus, Ohio, U.S.A.
FIELDS OF STUDY
Major Field: Dairy Science
Studies in Physiology: Professors ft; R. Gomes, H. C. Hines, T. M. Ludwick, N. L. VanDemark, and ft. G. Venzke
Studies in Biochemistry: Professors D. H. Ives, M. J. Klapper, R. 0. Moore and G, S. Serif
iVi.3 TABLE OF CONTENTS
Page ACKNOWLEDGMENTS ii
VITA ...... iv
LIST OF TABLES ...... vi
LIST OF ILLUSTRATIONS
INTRODUCTION o..*...... o 1
Chapter
I. REVIEW OF LITERATURE ...... 3
Occurrence of Capacitation in Various Species of Animals In Vivo Capacitation In Vitro Capacitation Inter-Specific Capacitation Nature of Capacitation Methods for Hyaluronidase Assay
II. MATERIALS AND METHODS ...... 18
General Procedures Experimental
III. RESULTS AND DISCUSSION ...... 30
IV. SUMMARY AND CONCLUSIONS...... 62
LITERATURE CITED ...... 64
v LIST OF TABLES
Table Page 1. Occurrence of Capacitation in Several Species of A n i m a l s ...... 5
2. Optical Density at 640 m/i of Various Quantities of Standard Hyaluronidase Solution upon Turbidimetric Assay ...... 22
3. Hyaluronidase Activity (H) and Motility Index (M) of Bull Semen (Observed at Different Intervals) upon In Vitro Incubation Under Different Treat ment sTT wo Levels of Antibiotic and Two Tempera tures of Incubation)...... 31
4.' Least-Squares Analysis of Variance for Hyaluronidase Activity of Bull Semen Under Different Treatments (Two Levels of Antibiotic and Two Temperatures of Incubation ...... 35
5. Hyaluronidase Activity Differences Between Pairs of Treatments (Two Levels of Antibiotic and Two Temperatures of Incubation) on Bull Semen, after Ignoring Time and Replications, Analyzed by *t' Test ...... 35
6. Hyaluronidase Activity (H) and Motility Index (M) of Bull Semen (Observed at Different Intervals) upon In Vitro Incubation at 38°C, 26°C, and 4 ° c ...... 38
7. Least-Squares Analysis of Variance for Hyaluroni dase Activity of Bull Semen upon Incubation at 38°C, 26°C, and 4 ° C ...... 41
8. Hyaluronidase Activity Differences Between Pairs of Treatments (38°C, 26°C, and 4°C) on Bull Semen after Ignoring Time and Replications, Analyzed by 't* Test ...... 41
9. Hyaluronidase Activity of Bull Semen with Respect to Motility Index upon In Vitro Incubation at 38°C, 26°C, and 4°C ...... 43
vi Table Page 10. Hyaluronidase Activity (H) and Motility Index (M) of Bull Semen at Four Intervals upon J[n Vitro Incubation with Rabbit Uterine Fluid, (3-Amylase, and KRPF Solutions at 38°C ...... 45
11. Least-Squares Analysis of Variance for Hyaluroni dase Activity of Bull Semen, at 38°C, Under Four Treatments Involving Use of Rabbit Uterine Fluid and 3-Amylase Solution ...... 47
12. Least-Squares Analysis of Variance for Hyaluroni dase Activity of Bull Seminal Plasma, at 38°C, Under Four Treatments Involving Use of Rabbit Uterine Fluid and 3-Amylase Solution ...... « 47
13. Hyaluronidase Activity Differences Bet. Pairs of Treatments Involving Use of Rabbit Uterine Fluid and 3-Amylase Sol. on Bull Semen, at 38°C, (Time and Replications Ignored) Analyzed by ’t* Test . 49
14. Hyaluronidase Activity (H) and Motility Index (M) of Rabbit Semen at Four Intervals upon In Vitro Incubation with Uterine Fluid, 3-Amylase, and KRPF Solutions at 3 8 ° C ...... • 50
15. Least-Squares Analysis of Variance for Variable Hyaluronidase Activity of Rabbit Semen, at 38°C (Observed at Four Intervals), under Four Treat ments Involving Use of Uterine Fluid and 3- Amylase Solution ....o,...... ooo. 52
16. Hyaluronidase Activity Differences Bet. Pairs of Treatments, Involving Use of Uterine Fluid and 3-Amylase Sol., on Rabbit Semen at 38°C, (Time and Replications Ignored) Analyzed by * tf T e s t ...... 52
17. Hyaluronidase Activity (H) and Motility Index (M) of Bull Semen upon Incubation in Rabbit Uterus, for Six Hours and upon In Vitro Incubation at 3 8 ° C ...... 56
18. Least-Squares Analysis of Variance for Variable Hyaluronidase Activity of Bull Semen upon Incubation (Six Hours) in Uteri of Estrous and Progesterone-Treated Rabbits ...... 57
19. Hyaluronidase Activity Differences Between Pairs of Treatments, Involving In Utero and In Vitro (38°C) Incubation of Bull Semen (Replications Ignored), Analyzed by *t' Test ...... 57
vii Table Page 20. Hyaluronidase Activity (H) and Motility Index (M) of Rabbit Semen upon Incubation in Rabbit Uterus for Six Hours and upon In Vitro (38°C) Incubation • . 58
21. Least-Squares Analysis of Variance for Hyaluroni dase Activity of Rabbit Semen upon Incubation (Six Hours) in Uteri of Estrous and Progesterone- Treated Rabbits ...... 59
22. Hyaluronidase Activity Differences Between Pairs of Treatments, Involving In Utero and In Vitro (38°C) Incubation of Rabbit Semen (Replications Ignored), Analyzed by *t* Test ...... 59
viii LIST OF ILLUSTRATIONS
Figure Page 1. Standard Reference Curve for Hyaluronidase Assay by Turbidimetric Method (National Formulary, 1965) ...... 23
2. The Relationship of Hyaluronidase Activity (Av. of Two Replications) of Bull Semen with Time Under Different Treatments (Two Levels of Antibiotic and Two Temperatures of Incubation) oee«e»eeoo«o««v«9 33
3. The Relationship of Hyaluronidase Activity of Bull Semen with Period of Incubation at 38°C, 26°C, and 4 ° C ...... 40
4. The Relationship of Hyaluronidase Activity of Bull Semen with Motility Index at 38°C, 2S°C and 4 ° C ...... 44
5. The Relationship of Hyaluronidase Activity of Bull Semen with Time upon Incubation with Rabbit Uterine Fluid (Heated and Unheated), f3-Amylase, and KRPF Solution at 38°C »••.«»• 46
6. The Relationship of Hyaluronidase Activity of Rabbit Semen With Time Upon Incubation With Uterine Fluid (Heated and Unheated), p- Amylase, and KRPF Solution at 38°C ...... 51
ix INTRODUCTION
It was demonstrated eighteen years ago that rabbit spermatozoa undergo certain physiological changes in the female reproductive tract before they are capable of penetrating the zona pellucida for fertili zation. This phenomenon was later called 'capacitation'. Evidence for capacitation has since been observed in some other species of animals.
The changes which take place in the spermatozoa in the female genital tract as a concomitant with capacitation remain still somewhat of an enigma. In rodents, changes in acrosome morphology (sometimes called 'acrosome reaction') have been seen following capacitation, while in rabbit no morphological changes in the capacitated spermatozoa have yet been clearly established. In cattle, the occurrence of capacitation is still a matter for debate.
The biochemical changes occurring as a result of capacitation are still more uncertain than the physiological principle itself. It has been suggested that release of hyaluronidase from spermatozoa occurs during capacitation. Also, acrosomes have been shown to be a rich source of hyaluronidase. It appears that capacitation may involve a series of step-wise biochemical changes, and it is possible that hyaluronidase may be one of the enzymes released by the spermatozoa upon incubation in the female genital tract, even if release of hyal uronidase alone may not be enough to give spermatozoa the capacity to
1 2 penetrate the egg.
No systematic study regarding the rate of release of hyaluroni dase during capacitation seems to have been reported. The present study was undertaken, therefore, to determine if any significant release of hyaluronidase occurs during conditions simulating capacita tion of rabbit spermatozoa. A parallel study was conducted on bull spermatozoa. REVIEW OF LITERATURE
Several reviews on capacitation of spermatozoa (10, 14, 17, 33,
55, 58, 73, 136) and on the role of hyaluronidase during fertilization of the egg (12, 13, 16, 47, 109, 120, 127, 128, 176) have been
published from time to time. Bibliographies regarding capacita-I ion
(5) and hyaluronidase (84) are also available.
Until 1951, the tacit assumption had been made that spermatozoa
ejaculated by fertile male mammals are fully prepared for entry into
the ovum. In that year, however, it was experimentally shown inde pendently by Austin and Chang (49) that rat and rabbit spermatozoa require incubation in the female genital tract for a period of several hours before they may penetrate and achieve syngamy with ova. This functional change in spermatozoa was termed ’capacitation' by Austin (9).
Austin (17) has defined 'capacitation* as a term:
used to denote a physiologic change that spermatozoa are believed to undergo as a necessary preliminary to penetration into eggs. The change normally occurs in the female genital tract during the transit of the spermatozoa to the site of fertilization in the ampulla of the fallopian tube and their stay there, pending the arrival of the egg.
Occurrence of Capacitation in Various Species of Animals
The need for capacitation of spermatozoa has been reported in several species of animals. The time required for capacitation differs
3 4 from species to species. The information available about some species along with important references has been summarized in Table 1.
No evidence has been reported for any species that spermatozoa, fresh from the male tract , can rapidly penetrate the eggs without undergoing a period of capacitation (136).
Dukelow e_t al. (71) advanced a hypothesis that the presence of decapacitation factor (DF) may be indicative of the need for capacita tion by that species. They have shown that seminal plasma of bull, rabbit, boar, stallion, and monkey (Rhesus) have DF activity, while dog, rooster, and human seminal plasma are devoid of it. Later, however,
Dukelow and Chernoff (68) confirmed the presence of DF in human seminal plasma also. This hypothesis appears to be in almost complete agreement with the information shown in Tdble 1 regarding capacitation in the several species studied so far.
In Vivo Capacitation
Rate of capacitation may vary in different environmental situa tions; rabbit spermatozoa, for instance, become capacitated within six hours in the estrous uterus, but require at least ten hours to reach this state in the Fallopian tubes (4). Dziuk (75) reported that spermatozoa which have resided in the female genital tract for a longer time have a better chance of fertilizing the eggs than those which have resided for a short time. Capacitation may be accomplished in the uteri of immature or ovariectomized animals (54, 137), and in the Fallopian tubes under progesterone domination (54) but evidently will not occur 5
TABLE X
OCCURRENCE OF CAPACITATION IN SEVERAL SPECIES OF ANIMALS
Species of Time Required References Animals (hours)
Cattle* Trimberger (180). Uncon firmed by Mahajan and Menge(117)
Ferret Chang and Yanagimachi (62)
Frog Shaver (161)
Hamster Chang and Scheaffer (61); Strauss (173)
Human* Dukelow and Chernoff (68)
Monkey (Rhesus) Dukelow and Chernoff (68); Dukelow et al. (71)
Mouse Braden and Austin (43)
Rabbit Austin (8); Chang (49, 52)
Rat Austin (51); Austin and Braden (21)
Rooster* Marion and Williams (121)
Sheep Mattner (125)
*Not clearly established. 6 within twelve hours in the progesterone-dominated uterus (54). Since rabbit spermatozoa may be capacitated successfully in the oviduct of the progesterone-treated or pseudopregnant female, Bedford (33) theorized that this served to. demonstrate the differential response of the uterus and oviduct to the circulating hormones. He further stated that it was probable that the failure of capacitation in the pseudopregnant uterus results from a deficiency of some essential factor, rather than from the presence of some inhibitory substance, since the suspension of capacitated spermatozoa in concentrated flushings from the pseudopregnant uterus did not appear to affect their fertilizing ability,(35).
Capacitation of rabbit spermatozoa was claimed to have been achieved in the isolated colon or bladder of male and female host rabbits, end in the anterior chamber of the eye and glandula vesicularis of male rabbits (137), but Hammer and Sojka (92) have since reported that rabbit spermatozoa could not be capacitated in bladder, colon, or knee joint.
In view of the results of Noyes _et al^, (137), it is evident that some phase of the capacitation process can occur in the absence of the secretions of the reproductive tract. It should be remembered, however, that in these experiments, the fertilizing ability of the j spermatozoa was assessed by the common method of transferring the j spermatozoa from the experimental environment into the oviducts of jI i the rabbits, at or soon after the time of ovulation; the experimental | I I spermatozoa would thus have spent some four to six hours in the I
Fallopian tubes before the termination of the fertile life of the ovum, I 7 which loses its integrity between six to eight hours (3, 51) after ovulation in rabbits. This allows the possibility that only partial capacitation may have been achieved in the bladder etc., the whole process being completed subsequently within the recipient Fallopian tubes.
The act of coitus has been reported to improve capacitation in the uterus (169). Soupart (167, 168) has further reported that administration of human chorionic gonadotrophin (HCG) produces the same result, with a highly critical dosage to response relationship; the peak effect was achieved with 75 I.U. The percentage of fertilized ova rose from 15.4, when no HCG was administered, to 74.5 when 75 I.U. of HCG were administered per animal. The fertilization of the ova was strongly inhibited at doses higher than 75 I.U.
In Vitro Capacitation
Most of the early attempts to achieve capacitation in vitro were not successful (52, 186). Recently, however, Kirton and Hafs (111) claimed to have obtained at least partial capacitation by incubation of rabbit spermatozoa in vitro in rabbit uterine fluid, and even in simple physiological solutions which contained p-amylase.
However, implications that it is simply contact with uterine fluid (or fluid in other effective sites) that causes capacitation, tend to be negated by the finding by Williams et al. (186) that spermatozoa placed in a millipore filter and left in the uterus failed to undergo capacitation. Moreover, Soupart and Orgebin-Crist (169) have reported 8 that capacitation did not take place in the uterus distended by double ligation. Studies by Barros (25) have indicated that ^-amylase does not play any role in the in vitro capacitation of hamster spermatozoa.
Capacitation of hamster spermatozoa can clearly take place in vitro in the presence of tubal secretions, since fertilization in vitro can be obtained with epididymal spermatozoa (25, 27, 191, 192, 193, 194).
There is a report too, that strongly supports the possibility of capacitation of rabbit spermatozoa in vitro in the presence of mucosal scrapings from the Fallopian tubes (164).
Recently, Ericsson (80) claimed to have capacitated rabbit spermatozoa upon incubation with mule eosinophils.
Inter-Specific Capacitation of Spermatozoa
Capacitation in the genital tracts of a number of different species is possible, as shown by the occurrence of a number of hybrids
(86) and observations on heterologous fertilization: rabbit X cotton-tail rabbit 8 (56, 60); hare 8 X rabbit ^ (2); sheep 8 X goat
£, and reciprocal (97); ferret £ X mink (f (57); rabbit 8 X snow-shoe hare <£, and reciprocal (59).
Inter-specific spermatozoon penetration not followed by fertili zation has also been recorded; rat spermatozoa in a Mastomys coucha
(= Rattus natalensis) egg (44), a hamster spermatozoon in a mouse egg
(26), and a rabbit spermatozoon in a rat egg (67).
The acrosome reaction of hamster spermatozoa recovered from the uterus 1.5-3 hours after mating was provoked when the spermatozoa were 9
incubated in vitro with mouse eggs (190) and mouse and rat tubal fluid
(27). Rabbit spermatozoa reportedly have been partially capacitated in
the rat uterus (37, 92) and even in dog uterus (92). Human and monkey
(rhesus) spermatozoa reportedly have been capacitated in the uteri of mouse, rabbit and hamster (68). Findings of Barros (26) support the
idea that hamster spermatozoa can achieve an advanced state of
functional capacitation in the tubal secretions of mouse and rat,
though participation of hamster secretions in the later stages of
capacitation cannot be absolutely excluded. He has advanced two tenta
tive hypotheses: (1) the active agent found in the mouse and rat is not identical with that found in the hamster and is less effective in
inducing capacitation; (2) the agent is the same but is present in
smaller amounts. In either case if will require a longer time to exert
its effect.
Nature of Capacitation
Possibly the first indication of the nature of the capacitation
changes came from the observation by Noyes et_ aL,. (137) that capacita
tion did not occur in spermatozoa which were held in a dialysis bag within the uterus of an estrous rabbit. This suggested that the change
involves a substance of high molecular weight, which is either con
tributed to the spermatozoa by the uterine environment and takes the
form of an enzyme or coenzyme, or else is removed from the spermatozoa
by the solvent action of the uterine fluid and has the property of a
protective or stabilizing coat on the surface of the spermatozoon. 10
Decapacitation Factor
The next significant step was Chang's (53) report that the
seminal plasma contained .substance(s) which appeared to abolish the
state produced by capacitation. Spermatozoa thus treated could regain
their fertilizing ability when placed in the Fallopian tubes before ovulation. The effective agent was called 'decapacitation factor' (DF)
(also called 'antifertility factor') and was further investigated by
Bedford and Chang (36) who found that it is non-dialysable, is precipitable by 50% ethar.ol, is stable to heating at 65°C for 30 min. , and can be concentrated by centrifugation at 105,000g for four hours.
They inferred that DF was of high molecular weight, possibly contained protein, and exerted its effect by interacting with an active system on the sperm head surface, thus blocking its reactivity to other sub strates. It was further surmised (183) that capacitation consists of the removal of DF, or something resembling DF, either by the action of enzymes in the genital tract secretions or (17) by the induction of a change in the electrostatic forces known to be involved in the formation and dissociation of some enzyme complexes. Later Weinman and Williams
(183), and Williams et al. (186) reported finding substances with strong DF activity in the epididymal fluid and inferred that DF is not the product solely of the accessory sex glands. Williams et al. (186) noted that DF was not destroyed by lecithinase, and neuraminidase.
Dukelow et al. (69, 70) found that it was resistant also to lysozyrae and pronase, and that dried preparations could be kept at 100°C for nine hours without undergoing destruction. Dukelow et al. (69, 71) and 11
Williams jet al, (184, 185) reported that DF can be inactivated by j3-amylase* p-amylase levels in the uterine fluids are about four times those detectable in blood serum (111)*
Acrosome Reactions and Release of Lytic Enzymes
It is clearly evident from the work on several marine inverte brates that in these organisms, spermatozoon entry depends upon the release from the sperm head of a lytic agent which digests a passage for the spermatozoon through egg investments (64, 65, 66, 182). The lysin is carried in the acrosomes and its release is brought about through the acrosome reaction, a specific rearrangement of the plasma and acrosome membranes. The normal stimulus for the acrosome reaction seems to be exerted by the substances emanating from the eggs and their investments, although the reaction may not be evoked until virtual contact is made between sperm head and the membrane on which lysin has to act.
Several pieces of evidence support the idea that the mammalian spermatozoon exhibits an acrosome reaction homologous in several respects with that in marine invertebrate spermatozoa (16, 63). When the mammalian reaction was first described (18, 19), it was regarded as a 'visible concommitant' of capacitation but the inadequacy of this view has since been apparent (4, 15, 29, 30, 31, 88). Austin and
Bishop (18, 19, 20) reported that in the golden hamster, Chinese hamster, and Libyan jird, living spermatozoa with reacted acrosomes were found in the Fallopian tubes, either in the vicinity of the eggs or within the egg membranes, but not in the uterus, where it could nonetheless be 12
surmised that many spermatozoa had undergone capacitation. Spermatozoa
which have attained the perivitelline space of the ovum exhibit acrosome
reaction in rabbit (129, 130), and in rat (139).
The acrosome reaction is much more reasonably considered as a
response to contact with egg investments or substances diffusing from
them. Recently Piko (143) has opined that the acrosome reaction seemed most likely to involve a specific interaction between receptor sites
(antifertilizin) on the spermatozoon head, which had been exposed as a
consequence of removal of the coating material during the process of
capacitation, and the complementary groups (fertilizin) around the egg.
The later might be the extensions of the egg surface that extend through
the zona into the cumulus, and it could be these processes, or substances
emanating therefrom, with which the spermatozoon first specifically
interacts. This interaction would create stresses in the plasma membrane of the spermatozoon that would result in the opening up of the acrosome. However Barros and Austin (27) are of the view that neither the egg nor its investments appear to be the source of significant amounts of any agent or agents responsible for acrosome reaction in hamster.
From the work of Fekete and Duran-Reynals (82), McClean and
Rowlands (126), Pincus (144, 145), Pincus and Enzmann (146, 148),
Schenk (157), Smith (163, 164) and Yamane (187, 188), it was suggested that role of hyaluronidase ('cumulus dispersing factor1) might be to disperse the cumulus cells surrounding the ovum to enable the spermatozoa to make contact with the zona pellucida. Soon afterwards, however, the 13 inadequacy of the concept that a high sperm concentration in the vicinity of the egg is needed to denude the ovum from the cumulus (147) became apparent from the experiments conducted by Austin (7), Austin and. Smiles (22), Bowman(42), Chang (48, 50), Leonard et al. (114), Lewis and Wright (116), Moricard and Bossu (131, 132), Odor and Blandau (138), and Wada et al. (181) who demonstrated that spermatozoa can, in fact, penetrate ova which are still enclosed in the cumulus. Shettles (162) reported that corona cells of human ova were partially denuded with fresh semen plus follicular fluid and completely denuded when pieces of tubal mucosa were also added, but that addition of hyaluronidase instead of tubal mucosa did not make any difference. The presumed neeid for hyaluronidase by the spermatozoa to 'burrow' through the intact cumulus (177) is Indicated by the reports of Austin (11). Austin (11) reported that if recently recovered hamster epididymal spermatozoa, which manifestly have unchanged acrosomes, are caused to swim through fresh medium before reaching a newly ovulated cumulus mass, they are quite unable to penetrate it. By contrast, cumulus masses recovered from mated animals often contain living spermatozoa with reacted acro somes and these freely make their way through the matrix. Recently
Bedford (33) reported similar findings in rabbit. Austin (17) stated that a false impression that unreacted spermatozoa are capable of penetrating the cumulus can arise from the failure to ensure that the fluid in which the spermatozoa were originally suspended does not reach the cumulus. In any suspension of spermatozoa, cells are dying and releasing hyaluronidase (107, 175), so that the suspension soon comes to contain a significant concentration of free enzyme. Acrosome 14 alterations occur either shortly before or immediately after death of spermatozoa (153).
The eggs.of birds, amphibia, and reptiles do not have cumuli, and there is a report that the testes of some of the species of birds, amphibia, and reptiles examined do not contain hyaluronidase (74).
Hyaluronidase is also absent in inactive testes like prepubertal and cryptorchid rabbit testes (171, 174). Moreover, morphological abnor malities of the acrosome have been reported to.be associated with partial or complete sterility in bulls (1, 40, 41, 95, 96, 98, 105,
151, 160, 178), boars (23, 24), and mice (102, 149). The role of hyaluronidase in cattle, however, becomes obscure because the small cumulus which the ovum has at the time of ovulation (154) is lost shortly after ovulation (123). Yamane (189) has shown that hyaluronic acid also exists in the ovum. He has suggested that the sperm cell carries hyaluronidase into the egg, and this breaks down the hyaluronic acid and allows the second polar body to be formed.
The role of hyaluronidase is also made apparent from the investi gation of several workers with hyaluronidase inhibitors. Martin and
Beiler (122) reported that administration of phosphorylated hesperdin, a hyaluronidase inhibitor, prevented conception in 44 out of 54 rats.
Similarly Parkes (141) reported success in the prevention of fertili zation by rabbit semen containing Trigentistic acid (rehibin). Parkes et al. (142) found that addition of a water soluble hyaluronidase inhibitor called '53D/k' to the semen strongly inhibits both hyaluroni dase and fertilization, yet in vitro, 53D/k had no adverse effect on sperm motility even in doses ten times as high as those used in the 15
semen used for insemination of the female rabbits. According to
Joel (106)s lowered hyaluronidase in semen accounts for some cases of human male sterility. On the contrary, some reports about increasing
the fertility of the semen by addition of hyaluronidase (6, 112, 113,
152, 165, 166) could not be confirmed by other workers (45, 46, 52, 83,
176). In fact, Sallman and Eirkeland (156) reported that the hyal uronidase titer of bull semen, beyond a threshold amount, had a high but negative correlation (-0.42) with the percentage of fertilization.
Johnston and Mixner (108) could not, however, establish any correlation between hyaluronidase content and fertility of bull semen.
A relationship between the mammalian acrosome reaction and the release of lytic agent or agents is indicated by several observations
(11, 16, 28). Austin (11) reported that the hamster spermatozoa
treated with dilute formalin solution were immobilized without detect able change in the acrosome. In addition, the induced fluorescence in these acrosomes after treatment with acridine orange closely resembled that of intact spermatozoa. Assay of the medium for hyaluronidase shotted no increase in concentration. On the other hand, when the spermatozoa were treated with dilute solutions of digitonin, the acro somes were visibly altered or detached, and no longer fluoresced, and the medium showed a tenfold increase in hyaluronidase content. There is, however, evidence that hyaluronidase is released from the spermatozoa info the seminal plasma when allowed to stand at room
temperature (101, 107, 123). There is a report that spermatozoa depleted of hyaluronidase are able to resorb it from the medium (77).
Bishop and Austin (38), Leuchtenberger and Schrader (115), and 16
Schrader and Leuchtenberger (158) suggested that hyaluronidase may be associated with the aerosome. Later, cell-free enzyme preparations consisting of lipoglycoprotein obtained from the acrosomes of ram and bull spermatozoa were shown to contain proteolytic and hyaluronidase activities (100, 170). Maneini et al. (118) also concluded from their immunofluorescent study that hyaluronidase is localized in the acrosome.
It has been suggested that capacitation is not an all or none phenomenon$ and probably involves more than one type of change (37).
Austin (17) propose4 that capacitation has the role of enabling the spermatozoa to undergo an acrosome reaction, which in turn causes the release of hyaluronidase, and that the enzyme makes it possible for the spermatozoon to pass through the cumulus. He further stated that potentiation of the spermatozoon for the acrosome may well depend upon the removal of an inhibitory or stabilizing polysachharide coat from the cell surface. A further evidence regarding the possible removal of a coat from the spermatozoal head during capacitation has been pre sented by Ericsson (78, 79) and Ericsson and Baker (81). They have * shown that a tetracycline HC1 binds to the surface of the spermatozoal head and it is specifically removed from the spermatozoal head upon capacitation, presumably because of the removal of the coat from the head.
Other Biochemical Changes
Another biochemical change reported in the spermatozoa which had been either incubated in utero or incubated in vitro in the genital fluids is the increased oxygen uptake in rabbit spermatozoa (85, 93, 94, 17
104, 133, 134), sheep spermatozoa (39, 85, 104, 150), swine spermatozoa
85, 159), and bull spermatozoa (140).
Methods for Hyaluronidase Assay
Several methods used for the assay of hyaluronidase have been reviewed by Meyer and Rapport (128) and Tolksdorf (179). The Tbrbidi- meteric method (179) in which results were expressed in 'Turbidity
Reducing Units', has been slightly modified by the American Pharma ceutical Association to express the results in 'National Formulary Units'
(135). Humphery (103) described the proposed International Standard for expressing the activities of hyaluronidase in 'International Units'.
All these units i.e. 'Turbidity Reducing Units' (T.R.U.), 'National
Formulary Units' (N,F. Units), and the 'International Units' (I.U.) are approximately equivalent to one another. MATERIALS AND METHODS
Virgin female rabbits about 6 to 8 months old, and male rabbits about a year old were obtained from a local breeder. All the rabbits were fed Purina rabbit chow ad. libitum.
General Procedures
Semen
Bovine and rabbit semen was used. In all cases, motility o£ the semen was observed under the microscope immediately after collec tion. The motility was graded according to Emmens (76):
Index of motility State of spermatozoa
4 Full activity, but there may be up to 30 or 40% of dead cells as in the fresh ejaculate 3^ A detectable damping of activity com pared with 4 3 Sluggish, rate of motion about two- thirds that of 4 2^ Most cells progressing but many stationary with tails vibrating 2 Most cells stationary, many with tails vibrating l|r Many motionless, none or almost none actively progressing 1 Hardly any motility, only tails moving (very rarely an occasional cell in actual motion) \ Only a few cells per field showing any movement 0 Completely motionless
Only semen showing motility index as 4 was used. The sperm
18 19 concentration was determined by hemocytometer. Semen was maintained at about 38°C until used. Krebs-Ringer-Phosphate-Fructose (KRPF) solution (119) used for dilution of semen was adjusted to pH 7.4 with
1 N HC1.
Bull semen was collected with an artificial vagina from a Brown
Swiss bull belonging to the Ohio State University. Semen was always used within 20 min. after its collection.
Rabbit semen was collected with an artificial vagina using an estrous female rabbit as dummy. Sometimes, if necessary, semen was filtered through a double layer of cheese cloth to remove a gelatinous clot from the semen. Semen was always used within 10 min. after its collection.
Uterine Fluid
The method described by Stevens e£ al. (172) was used for obtain ing uterine fluid from rabbits. The method consisted of ligating with synthetic suture, size 0, the uterus of an estrous female rabbit from the cervical as well as the tubal ends for a week to allow accumula tion of fluid. The fluid was aspirated with a 25-gauge needle attached to a syringe. The uterus was returned to normal position, and the skin wound was sutured as before, to allow fox1 further accumulation of fluid. The fluid from both horns and sometimes from more than one doe was pooled, centrifuged at 2,000g, 4°C for 10 min. The supernatant was treated with 500 I.U. of Pencillin per ml, and stored at 4°C. It was warmed to 38°C just before use.
Fluid from the uterus of an estrous cow was aspirated with a 20
pipette. The fluid was centrifuged at 109000g for 10 min. at room
temperature. The supernatant was further treated as described for
rabbits. The fluid was viscous, and it did not lend itself to accurate
measurements.
Heated uterine fluid was prepared by incubating uterine fluid
in a tightly stoppered test tube in a water bath at 70°C for 30 min.
Sampling Procedures
In experiments conducted under jLn vitro incubation, 0.05 ml
samples were drawn with 'Eppendorf' pipettes. A new tip was used for
each sample. The sample was transferred directly into a centrifuge
tube containing 0.95 ml diluent-for-hyaluronidase (135), mixed, centri
fuged at 10,000g, 4°C for 10 min. The supernatant was aspirated care
fully and stored at 4°C until assay for hyaluronidase activity was
carried out.
Hyaluronidase Assay
The Turbidimetric method (179) as described in modified form
in National Formulary (135) was used for the determination of hyal
uronidase activity in the samples.
The method consisted of incubating, at 37*0.5°C, 125 pz of
sodium or potassium salt of hyaluronic acid in 0.5 ml of buffer
solution with either 0.1, 0.2, 0.3, 0.4 or 0.5 ml of suitably diluted
enzyme solution containing undetermined amount of hyaluronidase. Except when 0.5 ml of enzyme solution was used, the total volume of solution
in each test tube, was made up to one ml by addition of appropriate
quantity of buffer. The rate of hyaluronic acid depolymerization 21 depends upon the amount of hyaluronidase present. After exactly 30 min., the reaction was stopped by addition of 4 ml of acidified horse serum.
Under these conditions the hyaluronic acid-protein precipitate forms a turbid suspension and can be measured in terms of its optical density.
After incubation with a suitable amount of hyaluronidase, less substrate is available for the interaction with protein and turbidity is reduced.
The method is capable of measuring graded responses. The turbidity was measured at 640 m/i with a Beckman spectrophotometer. Model DB.
Purified testicular hyaluronidase* was used as a reference standard. Hyaluronic acid^ extracted from human umblical cord, was used as substrate. A standard reference curve of optical density verus hyaluronidase activity was prepared with every new set of reagents for hyaluronidase assay. A typical standard reference curve is shown in
Figure 1 and the relative data are presented in Table 2.
Assays were conducted as soon after the end of experiment as possible but were always completed within 24 hours of the collection of the samples. The assay of each sample was repeated until two almost identical consecutive readings on duplicate samples were obtained within the linear range of the standard reference curve. The average of these two readings was used in calculating the hyaluronidase activity of that sample.
The hyaluronidase activity was expressed in terms of N.F. units
*Type IV, from bovine testes, containing 750 N.F. units per mg. supplied by Sigma Chem. Co., St. Louis, Missouri, U.S.A.
^Grade I, supplied by Sigma Chem. Co., St. Louis, Missouri, U.S.A. 22
TABLE 2
OPTICAL DENSITY AT 640 mju OF VARIOUS QUANTITIES OF STANDARD HYALURONIDASE SOLUTION UPON TURBIDIMETRIC ASSAY3
Hyaluronidase Log. of Optical Density Solution Substrate^ Diluent Serum Solution (ml) (ml) Replications (ml) Volume N.F. Mean (ml) Unitsc 1 2
0.5 0.5 0.75 0 .0 4.0 0.040 0.042 0.0410
0.5 0.4 0.60 0.1 4.0 0.083 0.084 0.0835
0.5 0.3 0.45 0 .2 4.0 0.146 0.147 0.1465
0.5 0 .2 0.30 0.3 4.0 0.236 0.236 0.2345
0.5 0.1 0.15 0.4 4.0 0.331 0.331 0.3310
aModified as in National Formulary (135).
^Contains 250 jug hyaluronic acid per ml.
cNational Formulary (N.F.) units of hyaluronidase contained in the respective volume of the solution. Log. Optical Density at 640E 0*10 0*05 0*30 0*25 0-20 0*15 0*35 FIGURE 1 STANDARD REFERENCE CURVE FOR HYALURONIDASE FOR CURVE REFERENCE STANDARD 1 FIGURE ylrnds ActivityHyaluronidase (N.R Units) - 0- -6 0 -4 0 0-2 SA YTRIIERCMTO (National METHOD TURBIDIMETRIC BY ASSAY omlr, 1965). Formulary, 0-8 23
24
per ml of the original undiluted semen or seminal plasma. Corrections,
necessitated as a result of dilution due to addition of antibiotics etc.,
were made.
Statistical Technique
The data were placed on computer cards and the IBM 360/75
computer was used for carrying out the computations. All analyses were based upon the 'least-squares and maximum likelihood general
purpose program' instructions for the use of which were written by
Harvey (99). Analyses of variance for hyaluronidase activity were
carried out by Least-Squares Analysis of Variance using unequal sub
class frequencies. In some cases, coefficients of correlation between
hyaluronidase activity and motility index of semen were calculated.
Differences in hyaluronidase activity of semen under various treat ments were also analyzed by using 't* test.
For most of the treatments, response curves of hyaluronidase activity versus period of incubation or motility, as the case may be, were drawn. The average of two or more replications was used for this purpose.
Experimental
The Relative Contribution by Spermatozoa and Microorganisms to the Hyaluronidase Content of Semen upon Jin Vitro Incubation
Two successive semen collections from the same bull were pooled. 25
Within twenty minutes of collections the semen was diluted with an equal volume of KRPF solution.
In each of the six test tubes, two ml of the diluted semen were added and incubated in a water bath at 38*0.5°C„ To two of the six tubes, selected at random, 1,000 I.U. of Penicillin per ml of the sample were added every 24 hours. To each of the remaining four tubes an equivalent volume of KRPF solution was added and two of these four tubes were immediately transferred to a water bath at 4°C. This was done in order to kill spermatozoa with a cold shock. The remaining volume of the diluted semen was centrifuged at 10,000g, 4°C for 10 min.
The supernatant was saved. Into each of two test tubes, 2 ml of the supernatant were poured, and the tubes were incubated in the water bath at 38*0.5°C. These tubes also contained a volume of KRPF solution equivalent to the volume of Penicillin added to the two tubes as described before.
At various intervals, samples were withdrawn from each test tube and assayed for hyaluronidase activity. Motility of semen was observed under the light microscope and recorded.
The Rate of Release of Hyaluronidase from Bull Spermatozoa upon In_Vitro Incubation at Different Temperatures
A single collection of semen from the bull was immediately diluted with an equal volume of KRPF solution containing 1,000 I.U. of
Penicillin per ml.
Diluted semen was equally divided into six test tubes. Ttoo of the tubes were randomly selected for incubation at 38°C, two at 26°C, 26
and the remaining two for incubation at 4°C. The temperature was
lowered @ 5 °C per hour.
Samples for hyaluronidase assay were withdrawn from each tube
at various intervals until sperm motility was reduced to almost zero.
Within five minutes of withdrawal of sampless the motility of semen in
each tube was observed under the light microscope and recorded.
The Rate of Release of Hyaluronidase from Bull Spermatozoa upon In Vitro Incubation with Uterine Fluids (Heated and Unheated) from Estrous Female Rabbits, Estrous Cows, and (3-Amylase Solution at 38°C
Two successive collections of semen from the bull were pooled and immediately diluted with an equal volume of KRPF solution contain
ing 1,000 I.U. of Penicillin per ml.
Half of the diluted semen was centrifuged at 10,000g at 4°C for
10 min. and the supernatant was saved.
The experimental group consisted of 12 test tubes each contain
ing 0.5 ml of the diluted semen. The control group also consisted of
12 test tubes each containing 0.5 ml of the supernatant obtained
following centrifugation of diluted semen. Randomly, two tubes from each group were selected for each of the 6 treatments i.e., rabbit uterine fluid, heated rabbit uterine fluid (70°C for 30 min.), cow uterine fluid, heated cow uterine fluid (70°C for 30 min.), p-amylase^
solution (0.1 mg per cent, prepared in KRPF solution), and KRPF
solution. To each tube, 0.5 ml of the respective fluid was added and
3Type I-B extracted from sweet potato, supplied by Sigma Chemical Co., St. Louis, Missouri, U.S.A. 27
all the tubes were Incubated In a water bath at 38*0.5°C.
At 0 9 2p 6 S and 10 hour intervals samples were withdrawn for
hyaluronidase assay. Motility of semen was observed under the light
microscope and recorded. The sampling from the tubes containing cow
uterine fluid had to be abandoned because the fluid was too viscous
to be accurately measured. Moreover, the semen did not uniformly mix
with the cow uterine fluid.
The Rate of Release of Hyaluronidase from Rabbit Spermatozoa upon In Vitro Incubation with Estrous Rabbit Uterine Fluid (Heated and Unheated), and (3-Amylase Solution at 38°C
One or more collections of semen were made with an artificial
vagina from one or more male rabbits to obtain a minimum volume of 1.5 ml
of semen. The semen was immediately diluted with an equal volume of
KRPF solution containing 1,000 I.U. of Penicillin per ml.
Diluted semen was equally divided into 8 test tubes. Two of
the tubes were randomly alloted to each of the 4 treatments i.e.,
rabbit uterine fluid, heated uterine fluid (70°C for 30 min.), p-amylase
solution (0.1 mg per cent, prepared in KRPF solution), and KRPF solution.
To each tube an equal volume of the respective fluid was added and they were incubated in a water bath at 38*0.5°C.
At 0, 2, 6 , and 10 hour intervals samples were withdrawn for
hyaluronidase assay. Motility of semen was observed under the light microscope and recorded. 28
The Release of Hyaluronidase from Bull and Rabbit Spermatozoa upon In Utero (Rabbit) Incubation
Four virgin female rabbits were kept under observation for three weeks. Each of two of these was injected intramuscularly with 25 mg crystalline Progesterone^ in corn oil, four days prior to in utero incubation of semen. The other two rabbits were considered to be in estrous if the vulva was found to be moist and reddish in color.
To anethetize the rabbits sodium pentobarbital solution (60 mg per ml) was very slowly administered intravenously into an ear vein until deep anesthesia was induced. The dose was found to be between two to three ml per rabbit. After shaving and sterilizing the abdominal area, a mid-abdominal, midline incision about five centimeter long was made, and the uterus was brought up through the incision. Each of the horns of the uterus was ligated with synthetic (Mersilene^) size 0, suturing thread, as tightly as possible, both at the cervical end, and at its junction with the oviduct, Intra-uterine injection was made just anterior to the cervical ligature with a 25-gauge needle attached to a half-ml syringe.
Semen from the bull and rabbit was used in separate trials.
After dilution of semen with an equal volume of KRPF solution, 0.1 ml of diluted semen was injected into each horn of the uteri of all four rabbits. The remaining diluted semen was equally divided into two test
^Supplied by Nutritional Biochemical Corporation, Cleveland, Ohio, U.S.A.
5Supplied by Ethicon Inc., Somerville, New Jersey, U.S.A. 29 tubes and incubated at 38°C. Immediately afterwards, samples were withdrawn from each test tube for conducting hyaluronidase assay, and motility of spermatozoa was recorded.
After 6 hours of intra-uterine injection of semen, all four rabbits were anaesthetized as before, the uteri were removed and rabbits were killed by an overdose of anaesthesia.
Horns of uteri were surgically separated in petri dishes, and blood etc. on the uterine horn was absorbed on a blotting paper. The
Fallopian end of the horn was placed inside the centrifuge tube while the other end was held up with an artery forceps. The Fallopian end was cut off just above the ligature, and contents were flushed with
1.9 ml of diluent-for-hyaluronidase (135), using a 22-gauge needle attached to a syringe, the needle piercing at about the same spot as that for the intra-uterine injection of semen. After removing the needle, any fluid remaining in the horn was squeezed out between the fingers. Motility of the recovered spermatozoa was observed under the light microscope and contents of the centrifuge tubes were centrifuged at 10,000g, A°C for 10 min, and the supernatant was saved for hyaluronidase assay.
Samples were also withdrawn simultaneously from the semen incubated in vitro and motility of semen was recorded. RESULTS AND DISCUSSION
The Relative Contribution by Spermatozoa and Micro organisms to the Hyaluronidase Content of the Semen upon In Vitro Incubation
From the data presented in Table 3 and illustrated in Figure 2, it is apparent that bacteria contribute relatively little to hyaluroni dase content of semen since there is a very slight increase in the hyaluronidase content of seminal plasma^even after 185 hours of incuba tion at 38°C. The slight increase in hyaluronidase content of seminal plasma may be from the few spermatozoa which were left after centri fugation and could be observed under microscope. These findings are in disagreement with those of Johnston and Mixner (107), who concluded from their study that most of the increase in the hyaluronidase con tent of semen after 24 hours incubation at 37°C was attributable to bacterial production of the enzyme. Sallman (155) reported that anaerobic conditions were highly favorable for hyaluronidase formation by streptococci. Since semen as well as seminal plasma was incubated under aerobic conditions, this could be one of the reasons for lack of significant contribution by bacteria to the hyaluronidase content of semen. The centrifugation of semen at 10,000g for 10 min. is expected to precipitate out a large population of the bacteria in
^Supernatant obtained upon centrifugation of the semen diluted with an equal volume of Krebs-Ringer-Phosphate-Fructose solution.
30 TABLE 3
HYALURONIDASE ACTIVITY3 (H) AND MOTILITY INDEX (M) OF BULL SEMEN (OBSERVED AT DIFFERENT INTERVALS) UPON IN VITRO INCUBATION UNDER DIFFERENT TREATMENTS (TWO LEVELS OF ANTIBIOTIC AND TWO TEMPERATURES OF INCUBATION)
Semen Seminal Plasma
•d o *rl With Without U Without Without Penicillin, &) fri to Penicillin, Penicillin, 4°C up to 137 hrs, & Penicillin,. U 38°C 38°C 38°C C 3 at 38°C thereafter o o •JJ ^ (0 JO Replications Replications 3 Replications Replications o 1-1 1 2 1 2 1 2 1 2
H M H M H M H M H M H M H H 0 3520 4 3420 4 3400 4 3470 4 3500 3 3370 3 1633 1613 6 16600 l| 16200 1§ 15880 1-| 16880 1| 8600 2 8600 2 1680 1700 15 18500 0 18350 0 18850 0 17750 0 11167 1 10900 1 2180 2180 21 19000 0 19600 0 19850 0 20500 0 11400 10667 | 2613 2647 27 19105 0 20212 0 20362 0 21971 0 11285 J 11061 § 2715 2695 33 19860 0 20362 0 21217 0 23228 0 11798 | 11966 $ 2682 2715 40 20212 0 20614 0 21720 0 24737 0 12569 | 12402 ! 2722 2594 50 20362 0 21167 0 26647 0 27502 0 12972 4 12904 § 2695 2715 60 20489 0 21602 0 32378 0 33592 0 13378 { 13490 | 2678 2678 TABLE 3 Continued
Semen Seminal Plasma
T3 •HO With Without Without Penicillin, Without u CU Penicillin, Penicillin, 4°C up to 137 hrs, & Penicillin, tu ^ (0 38°C 38 C at 38°C thereafter 38 C es u O 3 •H O ■u ja «8 Replications Replications Replications Replications $ O rs M 1 2 1 2 1 2 1 2
H M H M H M H M H M H M HH 70 20843 0 22007 0 33086 0 34098 0 13221 0 13727 0 2664 2678 80 22361 0 21753 0 34604 0 35615 0 13378 0 13378 0 2712 267S 90 22007 0 22361 0 39157 0 41180 0 13828 0 13378 0 2712 2678 115 22361 0 21753 0 40168 0 40978 0 13322 0 13727 0 2698 2678 137 22260 0 23018 0 27319 0 30860 0 13378 0 13558 0 2678 2813 144 13659 0 14064 0 150 18809 0 17370 0 160 20742 0 21501 0 172 33895 0 34604 0 185 21501 0 22108 0 18314 0 18820 0 39966 0 38448 0 3306 3339
aExpressed as N.F. units per ml of undiluted semen or seminal plasma. U3 ro Hyaluronidase Activity (N.F. Units per ml) 41,500 3 £00 2 11,500
1,500 fe £00 0 OF INCUBATION). OF SEMEN WITH TIME UNDER DIFFERENT TREATMENTS (TWO LEVELS OF ANTIBIOTIC AND TWO TEMPERATURES TWO AND ANTIBIOTIC OF (TWOLEVELS TREATMENTS DIFFERENT UNDER TIME WITH SEMEN
FIGURE 2 THE RELATIONSHIP OF HYALURONIDASE ACTIVITY (AV. OF TWO REPLICATIONS) OF BULL OF REPLICATIONS) TWO (AV. OF ACTIVITY HYALURONIDASE OF RELATIONSHIP THE 2 FIGURE S e m i n a l P l a s m a , w i t h o u t P e n i c i l l i n, 3 8 ° C 0306 / / S @ m e n , w i t h o u t P e n i c i l l i n . , 3 8 ° C S e m e n , w i t h o u t P e n i c i l l i n ,4°C S e m e n , w i t h P e n i c i l l i n,38°C Incubation Period Period (Hours) Incubation 0 9 120 137 38°C 150 -gsg! 180
34 addition to the spermatozoa. This procedure, however, did by no means render the seminal plasma sterile as was evident from the large number of bacteria that x^ere subsequently observed under the microscope. Sub sequent development of turbidity and flakiness in the seminal plasma was typical of bacterial growth. It is, nevertheless, possible that centrifugation process may have changed, to some extent, the distribu tion of different types of bacteria in the seminal plasma.
Results of analysis of variance for hyaluronidase activity, carried out on the data up to and including the 137 hour interval, are presented in Table 4. The data pertaining to the period beyond 137 hours were not considered for analysis since the temperature of one of the treatments was changed from 4°C to 38°C. Differences between treatments were found to be significant ( P < .01) while the differences between replications were not significant (P<,05). The differences between all pairs of treatments were found to be significant ( P < .01) by 't.' test (Table 5).
Although the design of the experiment did not lend itself to the study of interactions since that was not the objective, indications of interactions between spermatozoa and temperature, and between spermatozoa and bacteria are evident.
The difference between the two treatments at 38°C is probably due to difference in the size of bacterial population since Penicillin was involved in one treatment and not in the other. Although no attempt was made to count bacteria in the semen, the large difference in bacterial numbers in both treatments could be easily observed, with out staining, under phase-contrast microscope. Since bacteria by them- 35
TABLE 4
LEAST-SQUARES ANALYSIS OF VARIANCE FOR HYALURONIDASE ACTIVITY OF BULL SEMEN UNDER DIFFERENT TREATMENTS (TWO LEVELS OF ANTIBIOTIC AND TWO TEMPERATURES OF INCUBATION)
Source D.F. Sum of Squares Mean Squares F
Total Ill 12106197532.06 Treatments 3 8558357337.74 2852785779.25 171.39** Replications 1 4317893.58 4317893.58 0.26 Incubation Periods 13 1978915477.19 152224267.48 9.14** Error 94 1564606823.55 16644753.44
^Significant at one per cent level.
TABLE 5
HYALURONIDASE ACTIVITY DIFFERENCES BETWEEN PAIRS OF TREATMENTS (TWO LEVELS OF ANTIBIOTIC AND TWO TEMPERATURES OF INCUBATION) ON BULL SEMEN, AFTER IGNORING TIME AND REPLICATIONS, ANALYZED BY 't! TEST
Pairs of Treatments3 D.F. Difference Standard t bet. Means Error
One Minus Two 94 -6682.14 1090.37 6.128** One Minus Three 94 7606.25 1090.37 6.976** One Minus Four 94 16777.57 1090.37 15.387** Two Minus Three 94 14288.39 1090.37 13.104** Two Minus Four 94 23459.71 1090.37 21.515** Three Minus Four 94 9171.32 1090.37 8.411**
aOne=Semen, with Penicillin, 38°C; two=semen, without Penicillin, 38°C; three-semen, without Penicillin, 4°C; four=seminal plasma, without Penicillin, 38°C.
^Significant at one per cent level. 36 selves produced relatively insignificant quantities of hyaluronidase, as stated before, the higher level of hyaluronidase content in the semen which did not contain any Penicillin, is probably due to inter action between bacterium and spermatozoon. The bacteria may produce the effect with the help of their metabolic products, exoenzymes, or change in pH of the medium. This suggests the possibility of two loca tions of hyaluronidase in the sperm. Some hyaluronidase is transferred into the surrounding medium easily, and most of this transfer is completed in about 15 hours while some of it may be located far inside the cell and is not released until after bacterial action.
The release of hyaluronidase at 4°C is significantly lower
(P<.01) than that at 38°C. This is probably due to interaction between the spermatozoon and temperature. The difference in the bacterial numbers in the semen incubated at 4°C and that containing Penicillin at
38°C was not evident under the microscope, nevertheless, the possibility of difference in hyaluronidase activities of these two treatments being attributable to bacteria, non-sensitive to Penicillin, cannot be ruled out.
Figure 2 also shows an unexpected decrease of hyaluronidase activity after 115 hours of incubation in the semen without Penicillin at 38°C. This may probably result from the denaturation of hyaluroni dase due to change in pH of the medium, effect of bacterial metabolic products, and other unknown factors. 37
The Rate of Release of Hyaluronidase from Bull Spermatozoa upon In Vitro Incubation at 38°C, 26°C, and 4°c
The data are presented in Table 6 and illustrated in Figure 3.
Analysis of variance (Table 7) shows that the difference between replications is not significant (P<.05) while the treatments are significantly different ( P < .01) from one another. When the data were analyzed b$r 't' test (Table 8), the differences between two pairs of treatments i.e. 38°C and 26°C, and 26°C and 4°C, were found to be significant at one per cent level, while that between 38°C and 4°C was found to be significant at five per cent level.
Coefficients of correlation between hyaluronidase activity and motility index of bull semen, calculated within-time-within-replica- tions-within-treatments, within-replications-within-treatment (time ignored), within treatment (time and replication ignored), and overall
(time, replication and treatment ignored), fall between -0.89 and
-0.98. The overall correlation coefficient is -0.9653. This implies that hyaluronidase activity of living spermatozoa has a very high negative correlation with their motility index.
Some hyaluronidase was found to be always present in semen even when assay was carried out as early as 10 min. after ejaculation. It was physically impossible to reduce the time of assay to less than 10 min. but from extrapolation of the curve it appeared that som-’ hyal uronidase was always present in semen at ejaculation.
The data in Table 9 have been rearranged from Table 6 with respect to motility index, after ignoring replications and periods of incubation. Curves, depicting the relationship of hyaluronidase TABLE 6
HYALURONIDASE ACTIVITY3 (H) AND MOTILITY INDEX (M) OF BULL SEMEN(OBSERVED AT DIFFERENT INTERVALS) UPON IN VITRO INCUBATION AT 38°C, 26°C, AND 4°C
Incubation Temperatures (°C)
38 26 4
Replications Replications Replications Mean Mean Mean Incubation 1 2 1 2 1 2 Period Period (Hours) I
H M H M H M H M H M H M H M H M H M 0 1079 4 1124 4 1102 4 1044 4 1034 4 1039 4 1014 4 105 0 4 1032 4 1 1179 3f 1253 3| 1216 3f 2 1392 3 1387 3 1390 3 1129 3f 1232 3f 1180 3f 3 1688 2f 1748 2f 1718 2f 4 2040 2§- 2128 2§ 2084 2f 1420 3 1520 3 1470 3 1504 3 1526 3 1515 3 5 2168 2§ 2171 2f 2170 2f 6 2500 2 2520 2 2510 2 1608 3 1603 3 1608 3 7 2776 2 2760 2 2768 2 8 3233 2 3233 2 3233 2 1848 3 1928 3 1888 3 2920 2 3016 2 2968 2 9 3650 if 3720 if 3685 if 10 4150 if 4100 if 4125 if 11 4950 1 4920 1 4935 1 12 5670 f 5700 f 5685 f 2000 2f 2056 2f 2028 2f 3920 If 4096 If 400S If 16 2376 2f 2440 2§ 2408 2f 4256 If 4376 If 4316 ll 23 2800 2 3020 2 2910 2 4536 if 4520 1| 4528 if TABLE 6 Continued
Incubation Temperatures (°C)
38 26 os k B 9 O O •H Replications Replications Replications «■ Mean Mean Mean ,a x i 9 O O *H B U M
H M H M H M H M H M H M 27 3220 2 3450 2 3335 2 31 5050 1 4950 1 5000 1 33 3870 1^ 3910 1|- 3890 Ij 41 4400 4670 lj 4535 if 5200 1 5100 1 5150 1 50 5070 5120 1 5095 1 5300 1 5050 1 5175 1 58 5733 5360 | 5546 | 5456 1 5133 1 5294 1 72 6400 6000 | 6200 I 5800 f 5500 \ 5650 |
aExpressed as N.F. units per ml of undiluted semen.
w VO Incubation Period (Hours) aAverage of two replications.
FIGURE 3 THE RELATIONSHIP OF HYALURONIDASE ACTIVITY OF BULL SEMEN WITH PERIOD OF INCUBATION AT 38°C, 26°C, and 4°C. 41
TABLE 7
LEAST-SQUARES ANALYSIS OF VARIANCE FOR HYALURONIDASE ACTIVITY OF BULL SEMEN UPON INCUBATION AT 38°C5 26°C, AND 4°C
Source D.F. Sum of Squares Mean Squares F
Total 75 209090852.57
Temperatures 2 9644170.82 4822085.41 16.88**
Replications 1 84.21 84.21 0 .0 0
Incubation Periods 21 161675185.25 7698818.34 26.95**
Error 51 14568091.64 285648.86
^Significant at one per cent level.
TABLE 8
HYALURONIDASE ACTIVITY DIFFERENCES BETWEEN PAIRS OF TREATMENTS (38°C, 26°CS and 4°C) ON BULL SEMEN AFTER IGNORING TIME AND REPLICATIONS, ANALYZED BY 't! TEST
Pairs of Treatment D.F. Difference Standard t bet. Means Error
38°C Minus 26°C 51 1118.82 206.39 5.421**
38°C Minus 4°C 51 523.45 223.86 2.338*
26°C Minus 4°C 51 547.42 132.44 4.133**
^Significant at five per cent level.
**Signifleant at one per cent level. 42 activity with motility index, drawn on the basis of rearranged data in Table 9, are illustrated in Figure 4. Since the resemblance among the three curves is apparent, statistical proof for the same was con sidered unnecessary.
Unlike the report by Masaki and Hartree (123), the transfer of hyaluronidase from spermatozoa into surrounding medium at room tempera ture is not completed in six hours, but the transfer of hyaluronidase continues for more than two days. The transfer of the enzyme from live spermatozoa appears to be a function of the motility index. In other words, the dying or the dead spermatozoon cell lets the hyaluroni dase escape into the surrounding medium. Since the motility index is also highly correlated with the per cent of live or dead cells, only the index was considered.
The Rate of Release of Hyaluronidase from Bull and Rabbit Semen upon In Vitro Incubation with Rabbit Uterine Fluid (Heated and Unheated), (3-Amylase, and KRPF Solution at 38°C
The data regarding the bull and rabbit semen are presented in
Tables 10 and 14, respectively. All statistical analyses were done with respect to hyaluronidase activity and not motility index. Separate analysis of variance were carried out with respect to bull semen data and bull seminal plasma data.
Analysis of variance on the data pertaining to bull and rabbit semen (Tables 11 and 15) show that the treatments are significantly different (P<.01) from one another while replications are not (P<.05).
The analysis of variance on data pertaining to bull seminal plasma 43
TABLE 9a
HYALURONIDASE ACTIVITY15 OF BULL SEMEN WITH RESPECT TO MOTILITY INDEX UPON IN VITRO INCUBATION AT 38°C» 26°C, AND 4°C
Incubation Temperatures (°C)
Index 38 26 4
4 1102 (2) 1039 (2) 1032 (2)
3I J 2 1216 (2) 1180 (2)
3 1390 (2) 1655 (6) 1515 (2)
2—2 1990 (6 ) 2218 (4)
2 2837 (6) 3122 (4) 2968 (2)
3905 (4) 4212 (4) 4284 (6)
1 4935 (2) 5095 (2) 5155 (8) 1 2 5685 (2) 5873 (4) 5650 (2)
aData from Table 6 have been rearranged.
^Average of all observations after ignoring time and replications. The number of observations averaged are shown in parenthesis. Hyaluronidase Activity(N.F. Units per ml) 0 0 0 , 5 0 0 0 , 4 2,000 6,000 1,000 4 FIGURE 4 THE RELATIONSHIP OF HYALURONIDASE ACTIVITY HYALURONIDASE OF RELATIONSHIP THE 4 FIGURE aAverage of two replications. two of aAverage 3 OF BULL SEMEN WITH MOTILITY INDEX AT 38°C, AT INDEX MOTILITY WITH SEMEN BULL OF 26°C, AND 4°C. AND 26°C, Motility Index 2
44 U®C ' 38°C ; 26°C
0
45
TABLE 10
HYALURONIDASE ACTIVITY3 (H) AND MOTILITY INDEX (M) OF BULL SEMEN AT FOUR INTERVALS UPON IN VITRO INCUBATION WITH RABBIT UTERINE FLUID*p-AMYLASE, AND KRPF SOLUTIONS AT 38°C
y—s Semen Seminal Plasma CO b3 a o O JS Treatments fH W Replications Replications 4J Mean Mean •S5 c q3 1 2 1 2 M tu
H M H M H M H H H 0 1808 3— 1840 3— 1824 3— 939 1053 1046 Rabbit 2 2806 2-L 28802* 28432* 1029 1029 1029 1 ^21 ^21 Uterine 6 7740 2 8140 2 7940 2 1053 1027 1040 Fluid 10 11200 0 11120 0 11160 0 1059 1083 1071
3i Heated 0 1840 J3— 2 1760 J2 1800 J23— 992 1005 998 Rabbit 2 2896 2 2840 2 2868 2 1003 1060 1032 1 1 1 Uterine 6 8440 2 8300 2 8370 2 1059 1080 1070 Fluid 10 10930 0 11250 0 11090 0 1080 1080 1080
0 1808 3? 1840 J3— 2 1824 J23— 920 960 940 (3- Amylase 2 2616 3 2600 3 2608 3 1080 1029 1054 Solution 6 3000 2 3016 2 3008 2 1053 1032 1042 10 5933 1 6160 1 6046 1 1053 1032 1042
0 1588 3i 1720 3~ 1754 939 939 939 KRPF 2 24964 2560 4 9528 2i 1060 1060 1060 Solution** 6 2976 2“ 2896 2 2936 2- 1059 1013 1036 10 5427 1 5733 1 5580 1 1080 1045 1062 .
aExpressed as N.F. units per ml of undiluted semen or seminal plasma.
bKrebs-Ringer-Phosphate-Fructose solution. Rabbit Uterine Fluid Heated Rabbit Uterine Fluid /3 -Amylase Solution KRRF Solution0, Control ^
»•*'
0 2 6 10 Incubation Period (Hours)
aKrebs-Ringer-Phosphate-Fructose solution.
^Average of all observations for seminal plasma after ignoring replications and treatments.
cAverage of two replications.
FIGURE 5 THE RELATIONSHIP OF HYALURONIDASE ACTIVITY OF BULL SEMEN WITH TIME UPON INCUBATION WITH RABBIT UTERINE FLUID (HEATED AND UNHEATED), 3-AMYLASE, AND KRPF SOLUTION AT 38°C. 47
TABLE 11
LEAST-SQUARES ANALYSIS OF VARIANCE FOR HYALURONIDASE ACTIVITY OF BULL SEMEN, AT 38®C, UNDER FOUR TREATMENTS INVOLVING USE OF RABBIT UTERINE FLUID AND (3-AMYLASE SOLUTION
Source D.F. Sum of Squares Mean Squares F
Total 31 330526210.96 Treatments (T) 3 59106239.59 19702079.86 1491.614** Replications 1 41400.03 41400.03 3.134 Time (M) 3 219505250.59 73168416.86 5539.468** Interactions, TM 9 51675192.28 5741688.03 434.694** Error 15 198128.47 13208.56
^Significant at one per cent level
TABLE 12
LEAST-SQUARES ANALYSIS OF VARIANCE FOR HYALURONIDASE ACTIVITY OF BULL SEMINAL PLASMA, AT 38°C, UNDER FOUR TREATMENTS INVOLVING USE OF RABBIT UTERINE FLUID AND p-AMYLASE SOLUTION
Source D.F. Sum of Squares Mean Squares F
Total 31 67803.46 Treatments (T) 3 2951.84 983.95 1.125 Replications 1 148.78 148.78 0.170 Time (M) 3 43434.09 14478.03 16.557** Interactions, TM 9 8152.03 905.78 1.036 Error 15 13116.72 874.45
**Significant at one per cent level 48
(Table 12) shows that neither treatments nor replications are signifi cantly different (P Both the treatments (heated and unheated uterine fluid) showing higher hyaluronidase activity were also accompanied by a faster decrease in motility index compared with the other two i.e. (3-amylase and KRPF solutions. Since motility has a high negative correlation with hyal uronidase activity, at least part of the difference in the hyaluroni dase activity of two groups of treatments, mentioned above, can be explained by the difference in the respective motility indices. The present data are not sufficient to estimate the percentage of the difference in hyaluronidase activities which can be explained by the differences in motility indices. The faster decrease in motility of spermatozoa in uterine fluid may be a consequence of increased metabolic activity of spermatozoa. TABLE 13 HYALURONIDASE ACTIVITY DIFFERENCES BET. PAIRS OF TREATMENTS INVOLVING USE OF RABBIT UTERINE FLUID AND 0-AMYLASE SOL. ON BULL SEMEN, AT 38°C, (TIME AND REPLICATIONS IGNORED) ANALYZED BY 't' TEST Pairs of Treatments D.F. Difference Standard t bet. Means Error Unheated Uterine Fluid, Minus 15 -90.25 57.46 1.571 Heated Uterine Fluid Unheated Uterine Fluid, Minus 15 2570.12 57.46 44.729** ^-Amylase Solution Unheated Uterine Fluid, Minus 15 2767.25 57.46 48.160** KRPF Solution3 Heated Uterine Fluid, Minus 15 2660.38 57.46 46.300** ^-Amylase Solution Heated Uterine Fluid, Minus 15 2857.50 57.46 49.730** KRPF Solution f}-Amylase Solution, Minus 15 197.12 57.46 3.430** KRPF Solution **Significant at one per cent level. aKrebs-Ringer-Phosphate Fructose solution. TABLE 14 HYALURONIDASE ACTIVITY3 (H) AND MOTILITY INDEX (M) OF RABBIT SEMEN AT FOUR INTERVALS UPON IN VITRO INCUBATION WITH UTERINE FLUID, p-AMYLASE, AND KRPF SOLUTIONS AT 38°C Incubation Replications Treatments Period Mean (hours) 1 2 H M H M H M 0 146 3f 152 149 3| Rabbit 2 182 2 190 2 186 2 Uterine 6 294 § 297 § 296 i Fluid 10 498 0 478 0 488 0 Heated 0 146 3| 144 3| 145 3| Rabbit 2 183 2 187 2 185 2 Uterine 6 306 | 296 | 301 \ Fluid 10 502 0 500 0 501 0 0 142 4 140 4 141 4 p-Amylase 2 169 3 179 3 174 3 Solution 6 275 | 289 | 282 \ 10 318 0 302 0 310 0 0 141 4 145 4 143 4 KRPF 2 190 3 170 3 180 3 Solution^ 6 . 283 | 281 | 10 334 0 326 0 330 i 0 aExpressed as N.F. units per ml of undiluted semen ^Krebs-Ringer-Phosphate Fructose solution. saaarasigiBwwaMn 51 5 0 ' O r Rabbit Uterine Fluid Heated Rabbit Uterine Fluid -Amylase Solution (0-lmg%) KRPF Solution0 400 E L- d> a. a> c Z> 2 300 >■ o < a> w o ■ o ‘c | 200 o >» X 100 Incubation Period (Hours) aKrebs-Ringer-Phosphate-Fructose solution kAverage of two replications. FIGURE 6 THE RELATIONSHIP OF HYALURONIDASE ACTIVITY OF RABBIT SEMEN WITH TIME UPON INCUBATION WITH UTERINE FLUID (HEATED AND UNHEATED), p-AMYLASE, AND KRPF SOLUTION AT 38°C„ 52 TABLE 15 LEAST-SQUARES ANALYSIS OF VARIANCE FOR VARIABLE HYALURONIDASE ACTIVITY OF RABBIT SEMEN, AT 38°C (OBSERVED AT FOUR INTERVALS), UNDER FOUR TREATMENTS INVOLVING USE OF UTERINE FLUID AND 3-AMYLASE SOLUTION Source D.F. Sum of Squares Mean Squares F Total 31 399601.46 Treatments (T) 3 21100.59 7033.53 131.473** Replications 1 34.03 34.03 0.636 Time (M) 3 336485.59 112161.86 2096.565** Interactions, TM 9 41178.78 4575.42 85.525** Error 15 802.47 53.50 **Significant at one per cent level. TABLE 16 HYALURONIDASE ACTIVITY DIFFERENCES BET. PAIRS OF TREATMENTS, INVOLVING USE OF UTERINE FLUID AND 3-AMYLASE SOL., ON RABBIT SEMEN AT 38°C, (TIME AND REPLICATIONS IGNORED) ANALYZED BY 't' TEST Pairs of Treatments3 D.F. Difference Standard t bet. Means Error One Minus Two 15 -3.38 3.66 0.923 One Minus Three 15 52.88 3.66 14.448** One Minus Four 15 45.88 3.66 12.536** Two Minus Three 15 56.25 3.66 15.369** Two Minus Four 15 49.25 3.66 13.456** Three Minus Four 15 -7.00 3.66 1.912 aOne=unheated uterine fluid; two=heated uterine fluid; three= 3-amylase sol.; four=Krebs-Ringer-Phosphate-Fructose sol. V **Significant at one per cent level. 53 Increased oxygen uptake by spermatozoa when incubated in uterine fluid has been reported in several species of animals including rabbit (85, 93, 94, 104, 133, 134) and bull (140). Moreover, viability of capaci tated spermatozoa is greatly reduced (72, 168). Since partial capacitation of rabbit spermatozoa (111) and even complete capacitation of hamster spermatozoa (25, 26) can be obtained upon incubation in fluids from female genital tract, it can be postulated that increased release of hyaluronidase from spermatozoa in uterine fluid may be due to a biochemical change concomitant with capacitation. There is evidence (11) that hyaluronidase is needed by the spermatozoa to 'burrow* into the cumulus of the ovum. Decapacitation factor (DF) is present in rabbit and bull semen (71). DF coats the spermatozoal head, and capacitation may consists of its removal (183). It seems reasonable to propose that, while the release of hyaluronidase activity from dead cells or dying cells nujiy be due to loss of selective-perme ability of cell membrane, the removal of DF may result in release of hyaluronidase in the live spermatozoa. Bedford (31, 32) suggested that some change in permeability of the head membrane of the rabbit spermatozoon may occur after residence in the estrous uterus for several hours, p-amylase found in large quantities in uterine fluid (111) may be responsible for destroying DF as shown by Williams et al^ (184). But since heating the uterine fluid (70°C for 30 min.) did not affect the increase in hyaluronidase activity of semen, either p-amylase was not denatured (which is unlikely) or p-amylase was probably not involved in the process at all. The later view is further substantiated by the fact that a 0 .1 mg per cent solution of p-amylase did not show 54 an increase in hyaluronidase activity comparable to that of uterine fluid. Only in the case of bull semen, p-amylase treatment showed a slight but significant increase (P«<.01) in hyaluronidase activity compared with that of KRPF solution. It is, however, possible either that the 0 .1 mg per cent solution was not strong enough or uterine fluid may have factors, other than p-amylase, that are also involved in the phenomenon. Original design of experiment incorporated incubation of bull spermatozoa with estrous cow uterine fluid also. The semen did not mix uniformly with estrous cow uterine fluid. Moreover, samples could not be accurately measured as the cow uterine fluid was very viscous and stringy. The part of the experiment involving cow uterine fluid had, therefore, to be abandoned. The bull semen showed an increase in hyaluronidase activity when incubated in heated and unheated rabbit uterine fluid, similar to that of rabbit semen. Bedford (34) and Bedford and Shalkovsky (37) have suggested that capacitation is a two-step process, the first of which can be accomplished in a foreign uterine environment, whereas the second is species-specific. It can be speculated that partial capacitation of bull spermatozoa might have been achieved in rabbit uterine fluid. The need for capacitation of bull spermatozoa was indicated from the study of Trimberger (180). A more extensive and direct approach to solve the problem by Mahajan and Menge (117) did not yield any positive results. Presence of DF in bull seminal plasma is, according to Dukelow et al. (71) an indi cation of the need for capacitation. An increase of hyaluronidase activity upon incubation with (foreign) uterine fluid may be considered 55 a further indication of the need for capacitation of bull spermatozoa. The Release of Hyaluronidase from Bull and Rabbit Spermatozoa upon jfa Utero (Rabbit) Incubation The data regarding bull and rabbit semen are presented in Tables 17 and 20, respectively. Analyses of variance were carried out on the data excluding the results of in vitro incubation because the differences in the sub-class frequencies were found to be unadjustable. The analyses of variance (Tables 18 and 21) show that differences between rabbits, and differences among uterine horns were not significant at the five per cent level. The differences between treatments (i.e. estrous and progesterone- treated rabbits) were significant at the one per cent level with respect to bull semen and at the five per cent level with respect to rabbit semen. The data were further analyzed by ' t’ test (Tables 19 and 22). In both cases (bull semen and rabbit semen), difference between hyal uronidase activity of semen recovered from uteri of estrous rabbits after six hours incubation, and the initial hyaluronidase activity were not found to be significant at the five per cent level. The former was, however, significantly less (P<.01) than the hyaluronidase activity of semen incubated in vitro for six hours. The hyaluronidase activity of the semen incubated for six hours in the uteri of estrous as well as progesterone-treated rabbits was significantly less (P<.01) than that of the semen incubated in vitro at 38°C, for the same period of time. Greenberg et al. (87) reported 56 TABLE 17 HYALURONIDASE ACTIVITY3 (H) AND MOTILITY INDEX (M) OF BULL SEMEN UPON INCUBATION IN RABBIT UTERUS, FOR SIX HOURS AND UPON IN VITRO INCUBATION AT 38°C Rabbits or Replications*5 1 2 Incubation Treatments Period (hours) Left Right Left Right Uterine Uterine Uterine Uterine Horn Horn Horn Horn H M H M H M H M Untreated (Estrous) 6 3470 | 3550 | 3300 § 2750 1 Progestrone 6 6900 l| 7S00 1| 9000 1^ 8800 1| H M H M In Vitro 0 2667 3| 2447 3| 6 24750 l| . 26500 l| aExpressed as N.F. units per ml of undiluted semen. ^Replication applies to in vitro incubation. TABLE 18 LEAST-SQUARES ANALYSIS OF VARIANCE FOR VARIABLE HYALURONIDASE ACTIVITY OF BULL SEMEN UPON INCUBATION (SIX HOURS) IN UTERI OF ESTROUS AND PROGESTERONE-TREATED RABBITS Source D.F. Sum of Squares Mean Squares F Total 7 50407787.50 Treatments 1 47190612.50 47190612.50 71.408** Rabbits 1 567112.50 567112.50 0.858 Uterine Horns 1 6612.50 6612.50 0.010 Error 4 2643450.00 660862.50 **Signifleant at one per cent level. TABLE 19 HYALURONIDASE ACTIVITY DIFFERENCES BETWEEN PAIRS OF TREATMENTS, INVOLVING IN UTERO AND IN VITRO (38°C) INCUBATION OF BULL . SEMEN (REPLICATIONS IGNORED), ANALYZED BY ’t» TEST Pairs of Treatments® D.F. Difference Standard t bet. Means Error One Minus Two 8 -4857.50 546.16 8.894** One Minus Three 8 710.50 668.90 1.062 One Minus Four 8 -22357.50 668.90 33.424** Two Minus Three 8 5568.00 668.90 8.324** Two Minus Four 8 -17500,00 668.90 26.162** Three Minus Four 8 -23068.00 772.38 29.866** ®One = JLn utero incubation in estrous rabbits; two = in utero incubation in progesterone-treated rabbits; three = in vitro incubation at zero hour; four = in vitro incubation for six hours. ♦♦Significant at one per cent level. 58 TABLE 20 HYALURONIDASE ACTIVITY3 (H) AND MOTILITY INDEX (M) OF RABBIT SEMEN UPON INCUBATION IN RABBIT UTERUS FOR SIX HOURS AND UPON IH VITRO (38°C) INCUBATION Rabbits or Replications^ 1 2 Incubation Treatments Period (hours) Left Right Left Right Uterine Uterine Uterine Uterine Horn Horn Horn Horn H M H M H M H M Untreated (Estrous) 6 498 2 432 2 408 2 442 2 Progesterone 6 559 2 616 2 574 2 479 2 HM HM In Vitro 0 414 3| 437 3£ 6 878 | 908 i aExpressed as N.F. units per ml of undiluted semen, kApplies to in vitro incubation. 59 TABLE 21 LEAST-SQUARES ANALYSIS OF VARIANCE FOR HYALURONIDASE ACTIVITY OF RABBIT SEMEN UPON INCUBATION (SIX HOURS) IN UTERI OF ESTROUS AND PROGESTERONE-TREATED RABBITS Source D.F. Sum of Squares Mean Squares F Total 7 39302.00 Treatments 1 25088.00 25088.00 11.805*’- Rabbits 1 . 5100.50 5100.50 2.400 Uterine Horns 1 612.50 612.50 0.288 Error 4 8501.00 2125.25 ^Significant at five per cent level. TABLE 22 HYALURONIDASE ACTIVITY DIFFERENCES BETWEEN PAIRS OF TREATMENTS, INVOLVING IN UTERO AND IN VITRO (38°C) INCUBATION OF RABBIT SEMEN (REPLICATIONS IGNORED), ANALYZED BY 't' TEST Pairs of Treatments* D.F. Difference Standard t bet. Means Error One Minus Two 8 -112.00 30.54 3.667** One Minus Three 8 19.50 37.41 0.521 One Minus Four 8 -448.00 37.41 11.975** Two Minus Three 8 131.50 37.41 3.515** Two Minus Four 8 -336.00 37.41 8.982** Three Minus Four 8 -467.50 43.20 10.822** aOne = in utero incubation in estrous rabbitsj two ■ in utero incubation in progesterone-treated rabbits; three = in vitro incubation at zero hour; four = in vitro incubation for six hours. ^Significant at one per cent level. 60 similar findings. They concluded from their observations that spermatozoa either did not secrete hyaluronidase during their residence in the female genital tract, or if they did, the mechanism for the removal of the enzyme functioned at a much higher rate than its secre tion. The low hyaluronidase activity of the semen recovered from the uteri may be due to inhibition by blood which invariably got mixed in it during flushing of the uterine horn, eventhough the blood etc. on the uteri was carefully absorbed on a blotting paper earlier. Blood serum from several species of animals including rabbit, have been reported to reversibly inhibit hyaluronidase activity (91, 124). Hadidian and Plrie (91) have shown that inhibition is directly pro portional to the concentration of serum, and that the reversibility of inhibition is a function of concentration of serum and time. Over a period of several hours, only partial reversibility had been noticed. Reversibility of serum inhibition has been achieved with addition of snake venom (89, 90) and some proteolytic enzymes (90). The semen recovered from the uteri of estrous rabbits has significantly less (P«<.01) hyaluronidase activity than that recovered from the uteri of progesterone-treated rabbits. No definite explana tions are available to account for these differences. Probably the recovery of semen from progesterone-dominated uteri is greater since little phagocytosis of spermatozoa by leucocytes is expected in pro gesterone-dominated uterus (110). Bedford (30, 32) reported that while damaged as well as intact spermatozoa were ingested by leucocytes in the estrous uterus, only spermatozoa with damaged acrosomes and/or damaged head membranes were ingested to any extent in the pseudo- 61 pregnant uterus. He further suggested that some change occurs in the surface membrane of spermatozoa in the estrous uterus which renders the intact sperm head acceptable to uterine leucocytes. The rabbit spermatozoa are capacitated in estrous uterus in six hours (8, 49), but are not capacitated in progesterone-dominated uterus even after twelve hours (54). The conditions which are usually assumed to be associated with spermatozoan capacitation, and which were simulated in this study, do not appear to contribute greatly to the variability in hyaluronidase levels as determined by the current procedures. SUMMARY AND CONCLUSIONS Semen samples from bulls and rabbits were evaluated for hyal uronidase activity after being subjected to various environments which are commonly suspected of having some association with the phenomenon of capacitation of spermatozoa. Environments involved both in vivo and in vitro conditions and included various temperatures, time intervals and uterine secretions. The Turbidimetric method was used for hyal uronidase assay of semen or seminal plasma. By any method of comparison (volume, sperm concentration, incuba tion intervals, etc), rabbit semen contained only a fraction of that found in bull sperm. Incubation of bull and rabbit semen in heated or unheated uterine fluids resulted in a faster decrease in sperm motility and a greater release of hyaluronidase than incubation at 38°C in p-amylase or Krebs-Ringer-Phosphate-Fructose solution. Semen recovered after six hours from the uteri of estrous rabbits showed no significant increase in hyaluronidase activity over the initial values, but semen from the progesterone-dominated uteri showed a very significant (P<.01) increase. The increase in enzyme for semen incubated in vitro at 38°C was, however, greater than that held in the progesterone-primed uteri, for similar lengths of time. Slight contamination by blood of semen samples recovered from the uteri may 62 63 have reduced the enzyme values of such samples. Although bacteria appear to contribute somewhat to the hyaluroni dase levels of semen, such contribution is very minor compared with the amounts produced by the spermatozoa. More enzyme is released from semen held at 38°C compared with that held at 4°C. The release of hyaluronidase is significantly correlated with sperm motility. Enzyme values rise rapidly as motility decreases. The coefficient of correla tion between hyaluronidase values and motility of spermatozoa was -0.96, The current data would tend to indicate that levels of hyaluroni dase could be associated with some of the factors associated with pre paration of spermatozoa for fertilization, however, factors others than enzyme levels also appear to be involved. LITERATURE CITED (1) Aamdahl, J. 1951. Forandring i sperraienes kromatinsubstans og i galea capitis som arsak til sterilitet hos okse. Nord. Vet. Med., 3:102. (2) Adams, C. E. 1957. 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Acquisition de la capacite fertilisatrice des spermatozides (*capacitation") dans les voies genitales femelles. In: La Fonction Tubaire et ses Troubles, p. 22-27. Masson et Cie, Paris. (11) Austin, C. R. 1960. Capacitation and the release of hyaluroni dase from spermatozoa. J. Reprod. Fert., 1:310. (12) Austin, C. R. 1961. The Mammalian Egg. Blackwell Scientific Publication, Oxford. (13) Austin, C. R, 1961. Fertilization of mammalian eggs in vitro. Intern. Rev. Cytol., 12:337. 64 65 (14) Austin, C. R. 1963. Capacitation of spermatozoa (Section of chapter entitled 'Fertilization and Transport of the Ovum') In: Physiological Mechanisms Concerned with Conception. C. G. Hartman ed., p. 289-298. Pergamon Press, New York. (15) Austin, C. R. 1963. Acrosome loss from the rabbit spermatozoon in relation to entry into the egg. J. Reprod. Fert., 6:313. (16) Austin, C. R. 1965. Fertilization. Prentice-Hall, Inc., New Jersey9 U.S.A. (17) Austin, C. R. 1967. Capacitation of spermatozoa. Intern. J. Fert., 12:25. (18) Austin, C. R., and M. W. H. Bishop. 1958. Capacitation of mammalian spermatozoa. Nature, 181:851. (19) Austin, C. R., and M. W. H. Bishop. 1958. Some features of the acrosome and perforatorium in mammalian spermatozoa. Proc. roy. Soc. B, 149:234. (20) Austin, C. R., and M. W. H. Bishop. 1958. Role of the rodent acrosome and perforatorium in mammalian spermatozoa. Proc. roy. Soc. B, 149:241. (21) Austin, C. R., and A. W. H. Braden. 1954. Time relation and their significance in the ovulation and penetration of eggs in rats and rabbits. Aust. J. Biol. Sci., 7:179. (22) Austin, C. R., and J. Smiles. 1948. J. roy. micr. Soc., 63:13. (23) Bane, A. 1961. Acrosome abnormality associated with sterility in boar. Proc. Fourth Intern. Congr. Anim. Reprod., The Hague. p. 810. (24) Bane, A., and L. Nicander. 1966. Electron and light micro scopical studies on spermateliosis in a boar with acrosome abnormalities. J. Reprod. Fert., 11:133. (25) Barros, C. 1968. 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