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0022-5347/96/1561-0267$03.00/0 THE JOURNAL OF UROLOGY Copyright 0 1996 by AMERICAN UROLOCICAL ASSOCIATION, INC.

Vol. 156, 267-270, July 1996 Printed in U S A .

DETRIMENTAL EFFECT OF LEFT VARICOCELE ON THE REPRODUCTIVE CAPACITY OF THE EARLY HAPLOID MALE GAMETE

NIKOLAOS V. SOFIKITIS,* IKUO MIYAGAWA, PETER INCZE AND SANTINA ANDRIGHETTI From The Reproductive Physiology and N F Center, Department of Urology, Tottori University School of Medicine, Yonago, Japan

ABSTRACT

Purpose: We evaluated the fertilizing capacity of round spermatids recovered from varicocel- ized rabbits.

Materials and Methods: A left varicocele model was created in 5 rabbits (Group A). Four rabbits underwent a sham operation (Group B). After 3 months round spermatid nuclei were collected from t h e left testicle of each rabbit and injected into oocytes. Oocytes were cultured for 24 hours.

Results: The proportion of 2- to 4-cell stage embryos to the successfully injected oocytes was significantly lower in group A than in group B (p <0.05).

Conclusions: Our findings indicate a detrimental effect of varicocele on the fertilizing potential of round spermatid.

KEY WORDS: fertility, spermatids, spermatozoa, varicocele, rabbits

Early studies have demonstrated that round spermatid (RS) nuclei injected into hamster oocytes are able to form pronuclei and participate in syngamy.1 Recent studies have shown pregnancies after intact RS cells have been transferred into mouse oocytes.2 Full-term pregnancies in animal models with live births and successful mating of these normal offspring have also been reported with oop- lasmic RS nuclei injection (ROSNI) procedures.3~4 How- ever, these studies have all been conducted with RSs re- covered from healthy male animals. Results of ooplasmic injections of RS nuclei obtained from animals with testic- ular impairment have not yet been reported. If ROSNI procedures could be successfully performed in the human, they might serve as a novel treatment for male factor infertility due to nonobstructive azoospermia when elon- gated spermatids are not present or in cases of severe oligospermia with a high percentage of dead spermatozoa. However, human patients who could benefit from ROSNI may not have healthy, anatomically and physiologically normal RSs but may rather suffer from defects in spermat- ogenesis or spermiogenesis. Therefore, a study evaluating the fertilizing potential of the early haploid male gamete in subjects with testicular damage is clinically relevant. Since the presence of left varicocele is the major identifi- able cause of male infertility576 and the male gamete that has just completed the second meiotic division is the RS, we evaluated the reproductive capacity of RSs obtained from left varicocelized animals.

The rabbit was chosen as the experimental model because it proved to be an excellent animal for the investigation of the physiology of the early haploid male gamete.3v4 Moreover, induction of left varicocele in the rabbit model has been accompanied by most of the consequences of varicocele known to occur in the

MATERIALS AND METHODS

Operative procedure. Fifteen 6-month-old male white New Zealand rabbits were used (mature; 3,700 to 4,300 g.). Intra- venous sodium pentobarbital (30 mg./kg.; Abbott Laborato-

Accepted for publication December 19, 1995. * Requests for reprints: Reproductive Physiology and IVF Center,

Department of Urology, Tottori University School of Medicine, Yonago 683, Japan.

Presented at the 90th Annual Meeting of the American Urological Association in La5 Vegas, Nevada, 1995.

ries, Chicago, Illinois) was used for anesthesia along with heparin sulphate (120 Ukg.) for anticoagulation. The left testicular vein (LTV), the left renal vein, the vena cava and the left lumbar segmental vein (left lumbotesticular trunk) were exposed through a midline abdominal approach and the presence of any anatomical variations observed as previously described.7 The LTV drained into the left renal vein in 1 rabbit, into the vena cava in 2 animals and into the left lumbotesticular trunk in the remaining 12 rabbits. The left lumbotesticular trunk drained into the vena cava or renal vein after its junction with the LTV in 3 and 9 animals, respectively. Only the latter 9 rabbits were selected for these studies. Five rabbits (group A) underwent creation of a left varicocele. The left lumbotesticular trunk was partially li- gated by a %zero silk ligature just above its junction with the LTV to one-fourth of its original diameter. The choice of this method for varicocele induction is explained by the anatomy of the left testicular venous drainage system in the rabbit.7-9 Four rabbits (group B) underwent a sham operation: the left lumbotesticular trunk was dissected free of the surrounding tissues and a 3-2130 silk suture was passed behind it without ligation. Postoperatively, all animals received 250 mg. ampi- cillin intraperitoneally.

Fertility evaluation. Three months later the fertility poten- tial of each rabbit was assessed. Each male rabbit was placed in the same cage as 4 adult healthy female rabbits for 4 days. Mating was confirmed in all of the 4 female rabbits. The day of the first mating was counted as day 0. From day 30, the female rabbits were checked for parturition. A male rabbit was considered fertile if its matings resulted in a t least 1 pregnancy.

Semen evaluation. Twenty-one to 26 days after the end of the 4-day fertility evaluation, 5 semen samples (1 per day) were collected from each rabbit. Semen collection was achieved by using an artificial vagina. A doe was placed in the same cage with the buck. Each male was allowed 3 false mounts prior to collecting the ejaculate. Semen volume was recorded after liquefaction and the sperm concentration was determined by a standard hemocytometer. The percentage of motile spermatozoa was determined by counting more than 300 spermatozoa in randomly selected fields. An estimation of the qualitative motility (forward progression; motility grade) was obtained by grading the type of movement or aggressiveness as previously described for human spermato- zoa.10 Ten droplets were used for the sperm count and mo-

267

268 VARICOCELE AND ROUND SPERMATID

tility assessment for each semen sample and the respective averages were calculated for each sample. One droplet of each semen sample was put on the stage of a confocal scan- ning laser microscope, and morphometric parameters of more than 500 undisturbed nonstained randomly selected sperma- tozoa were calculated as previously described.10 The maximal length of the sperm head (MLH), the maximal width of the sperm head (MWH), the length of the midpiece (LMP) and the ratio of the acrosomal region of the sperm head (ARHS) to the total surface of the sperm head (TSH) were measured. The percentage of living spermatozoa was assessed in each semen sample by the trypan blue stain."

Testicular temperature 1 serum testosterone measurements. The day after the last semen sample was collected, the left and right testicular and intraabdominal temperatures were recorded as previously described.8.9 Testicular temperature was assessed by percutaneous insertion of a 29-gauge needle probe attached to a digital thermometer (Unique Medical, PTC 201 model, Tokyo, Japan). Intraabdominal temperature was monitored with a rectal probe and body temperature was maintained between 36.8 and 37.1C with radiant heat throughout this procedure. Two blood samples were aspi- rated from 2 ear veins of each rabbit to measure peripheral basal serum testosterone levels. Testosterone was measured in triplicate as previously described.8.9

Isolation of round spermatids. Three days after assessment of the testicular and intraabdominal temperatures a midline abdominal incision was performed and the maximal diameter of the LTV at the level of the ipsilateral inguinal canal was measured. Then, the left testicle was harvested from each rabbit and placed in erythrocyte- lysing buffer (155 mM. NH,Cl, 10 mM. KHCO,, 2 mM. ethylenediaminetetraacetic acid, pH 7.2). Following removal of the tunica albuginea, the seminiferous tubules were washed in Dulbecco's phosphate buffered saline (Sigma Chemical Company, St. Louis, Mis- souri) with the addition of 5.6 mM. glucose and 5.8 mM. sodium lactate (modified Dulbecco's phosphate buffered sa- line)3.4 and cut into small pieces with a pair of fine scissors. The cell suspension was flushed out through a 25 to 40 pm. mesh (Whatman filter, Tomoda Inc., Tokyo, Japan), the fil- trate was collected and centrifuged at 250 X g for 5 minutes, and the sedimented cells were resuspended in modified Dulbecco's phosphate buffered saline. The round spermatids could be easily identified by the presence of a single or mul- tiple large (acrosomic) granule adjacent to the nucleus as we previously described (figJ.3.4 The round spermatids were as- pirated via the micromanipulator pipette and transferred to a modified Dulbecco's phosphate buffered saline. The inter- nal diameter of the pipette was 20 to 30 pm. prepared by using a FlaminglBrown Micropipette Puller (P-97/IVF, Sutter Instruments, Novato, California). The percentage of living spermatids was assessed in each testicular sample by the trypan blue exclusion test." Some fractions of the recov- ered cells were observed via transmission electron micros- copy as we previously described.3.4 This confirmed that the aforementioned method for separating spermatogenic cells results in the final fraction being 98% to 99% RSs.3.4

Nuclei isolation. The selected RSs were transferred from modified Dulbecco's phosphate buffered saline in a hyper- tonic sucrose solution consisting of 50 mM. Tris, 10 mM. MgCl,, and 550 mM. sucrose, centrifuged a t 1,000 x g for 7 minutes and resuspended in the same hypertonic solution containing Triton X-100 (0.035%: v/v). After gentle pipettings for 20 seconds and further centrifugation at 1,OOOxg for 7 minutes, the naked nuclei in the pellet were collected and transferred into modified Dulbecco's phosphate buffered sa- line. Isolated nuclei were washed 3 more times in modified Dulbecco's phosphate buffered saline and finally resus- pended in the same medium supplemented with 1 mM. MgCl, and 4% polyvinylpyrrolidone (molecular weight 360,000, PVF' K 90, ICN Biochemicals, Costa Mesa, Califor-

Observation of rabbit round spermatid via confocal scanning laser microscope ( X 7,500). Arrow indicates granule adjacent to nucleus.

nia) and kept at 34C. Some of the selected nuclei were ob- served via transmission electron micro~copy.~.~ Twenty nu- clei per animal were processed for ooplasmic injections.

Isolation of oocytes. Healthy female white New Zealand rabbits 8 to 9 months of age underwent superovulation in- duction as previously described.3.4 Oocyte donors were sacri- ficed for ovum pick-up. After excision, the fallopian tubes were trimmed free of excess tissue and flushed with medium (modified RD medium) similar to the RD medium previously described in detai1.3.4 However, it contained Dulbecco's low- glucose modified Eagle's Medium (GIBCO Company, Grand Island, New York) mixed 1:l (vh) with RPMI 1640

Preparation of oocytes looplasmic injections of RS nuclei. One hundred and eighty oocytes were collected by the above method. The oocytes were washed 3 times in modified RD medium and freed from cumulus cells by treatment with 0.1% bovine testicular hyaluronidase (Sigma Chemical Com- pany, St. Louis, Missouri) in modified RD medium for 2 minutes at 37C. Cumulus free oocytes were rinsed with mod- ified RD medium and preincubated at 1OC for 2 hours in 5% CO, in air. Electrical stimulation was then applied with a single electrical pulse (1250 V/cm., 100 psec.) as previously reported.4 "he oocytes were then transferred into modified RD medium for injection. Round spermatid nuclei were in- jected into oocytes within 40 minutes of isolation. A single RS nucleus was injected into each oocyte as we previously de- ~ c r i b e d . ~ , ~ The injected oocytes were then transferred in mod- ified RD medium containing taurine 10 mM. and incubated at 37C under 5% CO, in air for 24 hours.

After incubation, the oocytes were observed under the in- verted microscope (Olympus M-70, Tokyo, Japan) for devel- opment of 2- to 4-cell stage embryos. All of these embryos were transferred to synchronized recipient healthy female rabbits who had received 75 IU of human chorionic gonado- tropin (hCG) approximately 24 hours earlier. Live offspring were counted and examined for any physical abnormalities.

The investigator performing the ROSNI procedures and incubations was blinded to the identity of the injected nuclei. All of the assays and the observations of the oocytes and embryos were similarly performed in a blinded fashion. Sta- tistical analysis was performed by using Wilcoxon's test and

(GIBC0).3*4

VARICOCELE AND ROUND SPERMATID 269

Chi-square test (Yates' correction). A probability of p <0.05 was considered statistically significant. Values were ex- pressed as means ? SD.

RESULTS

Testicular temperature I testosterone profiles / semen parame- ters lfertility potential. The mean left and right intraabdominal versus testicular temperature differences were significantly lower in group A (2.2 ? 0.2C and 2.3 t- O.lC, respectively) than in group B (4.1 t 0.1C and 4.0 t O.lC, respectively). There was no significant difference in the basal peripheral serum testos- terone levels between the 10 samples of group A (3.3 ? 1.2 ng./ml.) and the 8 samples of group B (2.9 ? 0.9 ng./ml.). Sperm concentration, percentage of motile spermatozoa, motility grade, and LMP were significantly lower in the varicocele group A than in the control group B (table 1). However, differences in the semen volume, MLH, MWH and 100xARHS/TSH ratio were not signfmmt. In all group A animals, significant dilation of the LTV (maximal diameter >4 mm.) was observed. The maximal diameter of the LTV in group B animals was <2 mm. All of the varicocelized rabbits were proven to be infertile. AU of the sham-operated rabbits were fertile and showed a mean number of offspring per mating equal to 9.3. Pregnancies were achieved in all of the 16 female rabbits mated with the 4 sham- operated animals. One hundred and forty-nine offspring were delivered.

Outcome of ROSNI procedures. There was no significant difference in the percentage of living RSs between groups A (93 ? 2) and B (91 t 2). Transmission electron microscopy of 33 and 52 nuclear sections prepared from control and vari- cocelized animals, respectively, revealed complete removal of the cytoplasm and the acrosomic granule and defects in the nuclear membrane compatible with the observations previ- ously reported.3.4 All of the successfully injected oocytes were activated (presence of at least 1 well-developed female pro- nucleus and 2 polar bodies3.4). At the end of the culture period, the proportion of 2- to 4-cell stage embryos to the number of successfully injected oocytes was significantly lower in group A than in group B (table 2). The proportion of offspring to transferred oocytes was significantly lower in group A than in group B. Live offspring appeared healthy and did not exhibit any gross abnormalities.

DISCUSSION

Our findings are consistent with previous data concerning the reduction of intraabdominal versus testicular tempera- ture difference and sperm concentration and motility in vari- cocelized rats, rabbits, dogs and monkeys, and humans with varicocele.12-21 In addition, the significantly shorter sperm

TABLE 1. Effects of varicocele on semen quality and sperm morphometry"

Group Varicocele (A) Control (B)

(nb = 5; kC = 25) (n = 4; k = 20)

Semen volume (ml.) Sperm concentration ( x 106/ml.) 7c motile spermatozoa Motility grade 1.3 f 0.4d % living spermatozoa MLH' (pm.) 7.3 f 0.4 MWH" (pm.) 4.3 t 0.3 LMF (urn.) 7.1 i 0.4d

1.8 f 0.4 146 f 43d 30 i 7d

89 f 5

1.9 f 0.4 313 2 36

84 f 5 3.3 2 0.2 92 2 3 7.5 f 0.3 4.2 f 0.3 7.8 i- 0.3

ioox ~ R H S R S H ~ 43.6 t 1.9 44.3 2 1.4

a Values are expressed as means f SD. Number of animals (n) within each group. ' Number of samples (k) within each group.

Within each line the superscript "dn indicates a significant difference (p 10.05).

MLH: Maximal length of the sperm head MWH: Maximal width of the sperm head; LMP Length of midpiece; 100XARHSA'SH: lOOX ratio of the acrosomal region of the sperm head to the total surface of the sperm head.

rABLE 2 . Effects of varicocele on round spermatid nucleus capacity for fertilization

Group Varicocele (A) Control (B)

(n" = 5) (n = 4)

Injected oocytes 100 80 Successfully injected oocytes 91 76 Oocytes arrested in pronuclear 70 (0.76Ib 13 (0.17)

stage plus first cleavage in progress (per successfully in- jected oocyte)

successfully injected oocyte)

bryo)

2- to 4-cell stage embryos (per 21 (0.23)b 63 (0.82)

Offspring (per transferred' em- 0 (0)b 6 (0.09)

Number of animals in each group. Within each line the superscript "bn shows significant difference (p 10.05). Only 2- to 4-cell stage embryos were transferred.

midpiece in animals with varicocele than in control animals may suggest that left varicocele affects the sperm cytoskele- ton. The LMP in human spermatozoa has been proven to be an important sperm morphometric parameter strongly and significantly correlated with sperm motility and fertilizing potential.10

The absence of any significant alterations in the peripheral serum levels of testosterone is consistent with previous find- ings in humans with left varicocele and left varicocelized rabbits.8.9.19 However, the absence of a significant difference in the basal serum testosterone level between the control and varicocele groups does not necessarily mean that there was no effect of the varicocele on Leydig cell function in terms of testosterone synthesis, because the actual peripheral se- rum testosterone levels often fail to indicate a small change in the rate of testosterone biosynthesis.22 A harmful effect of left varicocele on Leydig cell function in rats has been previ- ously reported.17-19

Infertility in the varicocelized rabbits may be attributable to the lower sperm concentration and motility and malforma- tions in the sperm cytoskeleton. These alterations may be due to defects in the spermatogenesis and epididymal sperm maturation process as a result of a secretory dysfunction of the Leydig and Sertoli cells. Leydig cells produce and secrete testosterone, which maintains the spermatogenic and epidid- ymal sperm maturation processes. Other studies have re- vealed anatomic or physiologic changes in the Sertoli cell in rabbits with left varicocele.s.21 Alterations in Sertoli cell function may contribute to the development of the detrimen- tal effect of varicocele on spermatogenesis and epididymal sperm maturation since Sertoli cells regulate testosterone concentration in the intraluminal compartment of the testi- cle and epididymis through the production of androgen- binding protein.

Ooplasmic ROSNI procedures in rabbits with varicocele resulted in fertilization, suggesting that some nuclei selected from RSs of varicocelized subjects have fertilizing capacity. The significantly lower values of the proportion of 2- to 4-cell stage embryos to the number of successfully injected oocytes after ROSNI procedures in the varicocele group compared with control animals indicates an adverse effect of left vari- cocele on the fertilizing potential of RSs selected from the ipsilateral testis. This difference in fertilizing capacity may not be attributable to differences in RS viability since there was no significant difference in the percentage of living sper- matids between the 2 groups. Such differences may be re- lated to an increase in left testicular temperature in varico- celized rabbits since previous studies of Nakamura and coworkers have indicated that increases in testicular temper- ature result in alterations in spermatid physiology.23 The above investigators have shown that incubation of RSs at a higher than normal testicular temperature leads to lower incorporation of amino acids into protein due to a slower

270 VARICOCELE AND ROUND SPERMATID

formation of the initiation complex 40s methionine tRNA. The significantly smaller ratio of the number of offspring to the number of transferred embryos and the absence of full- term pregnancies in the varicocele group indicate tha t em- bryos developed after fertilization of oocytes by RS nuclei recovered from animals with varicocele have an impaired potential for implantation. This can be directly correlated with the presence of varicocele in these animals.

We are certain that our methodology for RS nuclei prepa- ration resulted in isolation of nude nuclei. In two previous inve~t igat ionsz.~ and i n this study a total of more than 400 nuclei were observed via transmission electron microscopy, and no cytoplasmic material was found around the nuclei. This confirms that rabbit RS nuclei without any cytoplasmic covering and subsequently without centrosomic material can fertilize oocytes, which can further develop in v i v ~ . ~ . ~ The above observations and the artificial parthenogenesis in sev- eral mammalian female gametes could be interpreted as a challenge to the classical theory of centrosomes, suggesting tha t mammalian oocytes lose their centrosomes when they mature and that centrosomes are introduced into oocytes (with the exception of mouse oocytes) by the ~ p e r m a t o z o a . ~ ~ Therefore, these data tend to support the speculation of Mazia that when paternal centrosomic material is absent, novel maternal spindle organizing centers can be developed or previously denatured or nonfunctional maternal centro- somic material can undergo resuscitation after oocyte activa- t i ~ n . ~ ~

Although i t i s difficult to extend conclusions from animal studies to the human, the results of the present study allow us to theorize that RSs from men with primary testicular dysfunction have fertilizing capacity. If the above hypothesis is valid, ROSNI procedures may have a role in the treatment of hyper- or eugonadotropic nonobstructive azoospermia with maturation arrest at the RS stage. Recently 4 human preg- nancies have been achieved by ooplasmic injections of RS nuc1eF and 2 pregnancies by injections of intact RSs.27

In summary, our data show impaired fertilizing capacity of the nucleus of the early haploid male gamete in rabbits with defects in the spermatogenic process due to the presence of left varicocele.

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