Induction of Infertility in Male Rats by Treatment with Gonadotropin Antiserum During Neonatal Life1 2

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1 BIOLOGY OF REPRODUTION 2, (1970) Induction of Infertility in Male Rats by Treatment with Gonadotropin Antiserum During Neonatal Life1 2 BRUE D. GOLDMAN1 AND VIRENDRA B. MAHESH Department of Endocrinology, Medical ollege of Georgia, Augusta, Georgia Received January 20, 1970 The possible physiological role of gonadotropins during neonatal life was studied in the rat, using suitable antibodies to block the effects of endogenous gonadotropins soon after birth. Antiserum which was prepared by immunizing rabbits with ovine LH crossreacted with rat LH and FSH. Male rats which were injected with this antiserum on days 1-5 of life were tested for fertility when they were days old. These animals were found to be infertile as judged by their failure to impregnate normal females. Successful matings did not occur even after priming the adult males with a large dose of testosterone propionate. In contrast to the result of antiserum treatment on days 1-5, fertility was not affected by injecting normal rabbit serum on days 1-5 or antiserum on days The effect of administering gonadotropin antiserum on days 1-5 was partially prevented if either 10 IU HG or 0.2 mg testosterone propionate was given on day 1. The latter observation suggested that the sterilizing effect of the antiserum was related to its ability to prevent gonadotropin-dependent testicular androgen secretion during the first few days of life. Antiserum treatment in male neonates failed to prevent masculinization of the hypothalamic control mechanism for gonadotropin release. Also, treatment of female neonates with gonadotropin antiserum did not appear to interfere with the normal onset of ovulatory cycles. Neonatal rats release measurable amounts of luteinizing hormone (LH) and folliclestimulating hormone (FSH) into the blood (Goldman et al., in preparation). However, until the present it has not been clearly established that gonadotropins perform a physiological role during neonatal life. For example, while there is abundant evidence that the rat testis secretes androgen during very early life (Harris, 1964; Resko et al., 1968), little is known concerning the degree to which this secretory activity is gonadotropin-dependent. 1 The results reported here were presented in part at the Fifty First Meeting of the Endocrine Society, New York, June 27-29, This research was supported in part by NIH research Grant AM and by NIH Training Grant 5TL HD a Present address: Department of Anatomy, ULA School of Medicine, Los Angeles, alifornia The present experiments were designed to investigate whether endogenous gonadotropins might play some role during a discrete period spanning the first few days of postnatal life. Rats were treated with gonadotropin antiserum during days 1-5 or days 7-11 of life, and various aspects of reproductive function were investigated afterwards. Female rats were studied only with respect to the onset of ovulatory activity as determined by the development of corpora lutea. In antiserumtreated males the following parameters were studied: (1) Achievement of fertility as judged by mating tests in which adult males were paired with normal females and (2) sexual differentiation of the hypothalamic mechanism which controls the cyclic release of gonadotropin in normal females and which is permanently suppressed in normal males. 444

2 ROLE OF GONADOTROPIN IN NEONATAL MALE RAT 445 in parentheses. MATERIALS AND METHODS Antiserum. Gonadotropin antiserum was prepared by immunizing rabbits against ovine LH (NIH-LH- Sli) as described previously (Goldman and Mahesh, 1968). A common pool of antiserum, obtained from three rabbits, was used in all experiments. This material will be referred to as GTH-antiserum (gonadotropic hormone antiserum) since it was found to be capable of neutralizing the biological activities of both LH and FSH. The GTH-antiserum was tested for its ability to neutralize the biological activities of various mammalian gonadotropins. These tests were carried out by employing the ovarian weight augmentation assay (Steelman and Pohley, 1953) to test for anti- FSH activity and the ovarian ascorbic acid depletion assay (Parlow, 1961) to determine anti-lh activity. Various hormone preparations were incubated overnight at 4 with the antiserum, and the solutions were then injected into the assay animals. ontrol animals received unincubated hormones or hormones which had been incubated with normal rabbit serum (NRS). The hormones tested were as follows: ovine LH (NIH-LH-S11), ovine FSH (NIH-FSH-53), HG (Ayerst), rat pituitary homogenate, hamster pituitary homogenate, and rabbit pituitary homogenate. Experimental design. Adult rats were obtained from the Holtzman o. (Madison, Wis.) and were bred in the laboratory. The date on which female breeders showed a sperm positive smear was designated as day 1 of pregnancy. Litters were born on day 21 or 22. Day 22 was designated as day 1 of life for all litters. (This system, it was believed, would provide the greatest consistency for comparison of stages of development between litters.) Four experimental series were prepared, each series being composed of an experimental and a control group. Within each litter approximately half the offspring were placed on each of the treatments within an experimental series. The designs of the series were as follows: Series I ontrol (8) ml normal rabbit serum (NRS) was injected on days 1, 3, and 5 of life. Experimental (7) ml GTH-antiserum was injected on days 1, 3, and 5 of life. Series 2 ontrol (6)-O.075 ml NRS was injected on days 7, 9, and 11 of life. The number of male rats in each group is shown Experimental (6)-O.075 ml GTH-antiserum was injected on days 7, 9, and 11 of life. Series 3 Both experimental and control groups received ml GTH-antiserum on days 1, 3, and 5 of life. of ontrol (5)-0.1 ml saline was injected on day 1 life. Experimental (5)-b IU HG (dissolved in saline) was injected on day 1. Series 4 Both experimental and control groups received ml GTH-antiserum on days 1, 3, and 5 of life. ontrol (6)-0.1 ml corn oil was injected on day 1 of life. Experimental (8)-0.2 mg testosterone propionate (dissolved in corn oil) was injected on day 1 of life. All injections were administered subcutaneously. After weaning, males and females were separated and housed in groups of 4-8 animals per cage. Experimental and control animals were mixed within these groups. Females. Female littermates were sacrificed at days of age. The ovaries were examined macroscopically for the presence of corpora lutea, and the ovaries and uteri were weighed. Males. Male littermates were tested for fertility beginning when they were days old. Successive tests were administered to most animals (two tests in most cases), but no males were used beyond the age of 180 days. The fertility of the males was judged on the basis of their ability to impregnate receptive females. Each male was tested individually by being placed in a large cage with one or two normal (untreated) proestrous females. The females were left with the males until at least 11 AM of the day of estrus. Vaginal smears were examined for sperm on the morning of estrus. Smears were examined daily thereafter to ascertain whether pregnancy or pseudopregnancy had been induced. All females which failed to continue estrous cycles after exposure to males were laparotomized days following exposure and were examined for the presence of fetuses or resorption sites. Females which were scored as pseudopregnant were those that showed only diestrous smears for more than 8 consecutive days and in which no fetuses or resorption sites were found. Most males were tested twice in the manner described above, and the tests were separated by an interval of at least 5 days. No differences were apparent between the results of successive tests with individual males in any of the groups. Males from Series 3 were treated with testosterone propionate following the mating tests described

3 446 GOLDMAN AND MAHESH above. Each male received daily subcutaneous injections of 2.5 mg testosterone propionate in 0.2 ml propylene glycol. After 5-7 days of treatment mating tests were again carried out. In these tests, the injections of androgen were continued during the period of exposure to females. After being tested for fertility, the males from Series 1 were castrated. The right testis was weighed; the left testis was fixed in Bouin s solution, sectioned, and stained with hematoxylin and eosin for histological study. At the time of castration, ovaries from immature female rats (21-23 days of age) were grafted under the kidney capsule (two ovaries per recipient). Five weeks later the grafts were removed and prepared for histological examination. The tissue sections were studied with respect to development of follicles and corpora lutea. RESULTS haracterization of antiserum. By using various doses of gonadotropins it was found that 1 ml of the GTH-antiserum was able to completely neutralize the biological activity of approximately 340.tg ovine LH or 300.tg ovine FSH. The antiserum also neutralized both LH and FSH activities of rat and hamster pituitary homogenates and the LH activity of rabbit pituitary homogenate. (ross-reaction with rabbit FSH was not tested.) In marked contrast, the LH activity of HG was not detectably altered by incubation with the antiserum (2 IU HG incubated with 0.2 ml antiserum). Females: Maturation of reproductive tract. The results obtained at autopsy of females from Series 1 and 4 are summarized in Table 1. Antiserum treatment failed to prevent ovulation from occurring by age days. Thus, there was little, if any, delay in the time of the first ovulation, which in our laboratory normally occurs at days of age in this strain. The size of the ovaries and uteri of the antiserum-treated females was normal. Females which received testosterone propionate on day 1 had small ovaries without corpora lutea as late as days of age. Males: Fertility. Males which had been treated with NRS on days 1-5 of life successfully impregnated 68% of the females to which they were exposed (Fig. 1). Two pseudopregnancies were induced by these males. In contrast, the males which had been treated with GTH-antiserum on days 1-5 failed to induce normal pregnancies in any of 21 females. However, one female was found to be carrying one live fetus following exposure to one of the males, accounting for the 5% pregnancy figure shown in Fig. 1. These antiserum-treated males did induce six pseudopregnancies. When injection of the antiserum was delayed until days 7-11 of life, the treatment did not impair fertility (Fig. 1). When HG or testosterone propionate was administered TABLE 1 OVARIAN FUNTION IN RATS TREATED WITH GONADOTROPIN ANTISERUM ON DAYS 1-5 OF LHE Ave. No. with Age at body Ovarian Uterine corpora autopsy No. weight weight weight lutea/ Treatment (days) rats (g) (mg) (mg) total NRS days 1,3, and ± 6.Oa ± l9.la 5/5 Antiserum days 1,3, and ± ± /8 Antiserum days 1,3, and 5; oil day /2 Antiserum days 1,3, and 5; testosterone propionate day ± ± 6.1 0/7 a Mean and SE.

4 ROLE OF GONADOTROPIN IN NEONATAL MALE RAT 447 Treated on Treated on Days 7, 9 & 11 a. 0 a. E NRS Antiserum j Pseudopregnant Ps) 6 23 Pregnant P) I 0 V a. FIG. 1. Percentages of pregnancies and pseudopregnancies resulting from pairings of normal females with adult males which had received normal rabbit serum (NRS) or GTH-antiserum during neonatal life. The first number above each bar denotes the number of males used; the second number indicates the total number of females used. Each male was used in at least two separate tests. on day 1 to males receiving antiserum on days 1-5, the effect of the antiserum seemed to be partially prevented (Fig. 2). The results obtained with the respective control groups confirmed the sterilizing effect of early treatment with GTH-antiserum and revealed that administration of the vehicles (saline and oil) used to disolve HG and testosterone did not interfere with this effect. Treatment with testosterone propionate during adulthood failed to alter the results of mating tests involving males which had received GTH-antiserum on days 1-5 (Fig. 3). However, when males which had received antiserum on days 1-5 as well as HG on day 1 were primed with testosterone propionate the percentage of fertile matings by these animals was increased to a level that was not statistically different from that observed in normal rats. In the course of the various studies described above it was observed that females which were made pseudopregnant by antiserum-sterilized males often had a relatively small number of sperm in the vagina on the morning of estrus but never the large numbers of sperm almost invariably found in the vaginal smears of females which had mated with fertile males. Males: Testicular development. The weight of the right testis from eight adult males ( days of age) which had been treated Effects of HG & T.stost.,on. Propio.tate NRS in Rats Treated with Antiserum (Days 1.3,S) (Days i,3,s) IU IG a. byl O.yii ;2O 60 OIl.2.i o. ii 10y ii fl Ps.udopr.gnant (Pa) [ : #{149} FIG. 2. Results of mating tests involving male rats which had received GTH-antiserum on days 1-5 of life and either human chorionic gonadotropin (HG), testosterone propionate (TP), or the appropriate injection vehicle on day 1. At the far right the results obtained with NRS-treated males are reproduced for comparison.

5 448 GOLDMAN AND MAHESH with NRS on days 1-5 was 1.83 ±.29 g (mean ± SE). The right testis from six littermate males which had received GTH-antiserum on days 1-5 weighed 1.96 ±.24 g. The difference between these two groups was not statistically significant. Furthermore, examination of histological preparations failed to indicate any differences between the NRSand antiserum-treated rats in regard to development of testicular tubules or interstitial cells or with respect to spermatogenesis. Males: Hypothalamic control of gonadotro pin release. Ovaries were removed from immature rats and were transplanted into eight adult, castrated male rats which had received NRS on days 1-5 of life. Five weeks later these ovaries showed marked follicular development, but no corpora lutea were present. A similar result was obtained when ovaries were grafted in six castrated males which had been rendered infertile by treatment with GTH-antiserum on days 1-5 of life, with the exception that in a single male one small corpus luteum was found in one of the grafted ovaries. Ovaries were also transplanted to five adult, gonadectomized female rats. All these ovaries showed numerous large corpora within 3 weeks. Thus, the failure of corpora formation in the males did not appear to be due to faulty procedure or to poor acceptance of the grafts. DISUSSION Radioimmunoassay of LH and FSH in rats at 1-12 days of age has revealed surprisingly high levels of both hormones in the serum of neonatal females (Goldman et al., in preparation). Neonatal males had very low serum gonadotropin concentrations; however, within 1 day following removal of the testes, serum concentrations of both LH and FSH in 5-7-day-old males rose to approximately the same levels as those observed in adult males 1 day postcastration. The observation that gonadotropins are secreted by the neonate raises the question of the possible physiological significance of these hormones in the newborn. Following hypophysectomy of adult rats of either sex, the gonads atrophy and reproductive function ceases (Jacobsohn, 1966). These effects can be reversed by injecting gonadotropins in the hypophysectomized animal. However, since animals hypophysectomized during neonatal life can survive for only a short time, it has proved difficult to evaluate the possible effects of a gonadotropin deficiency in very early life. The present experiments indicate a convenient means to making such an evaluation since it was possible to temporarily block the effects of endogenous gonadotropins in neonates (by injection of GTH-antiserum) without interfering with normal body growth and viability. The failure of GTH-antiserum to interfere with development of the female reproductive tract or to prevent ovulation from occurring was not surprising since neither gonadotropins nor gonadal steroids are known to be necessary for neonatal differentiation of the femaletype hypophyseal-gonadal axis. Also, the anovulatory state which developed in female rats which were treated with testosterone propionate during neonatal life (Table 1) has been observed by others and appears to be due to permanent suppression of the hypothalamic center which controls the cyclic release of gonadotropins (Harris, 1964). In male rats antiserum treatment on days 1-5 of life resulted in reproductive sterility, characterized by an almost total failure to achieve successful impregnations in mating tests. This effect was absent when treatment was delayed until days The latter finding demonstrates that the effect of early treatment is not due simply to persistence of antibodies in the plasma of recipient males. The results also suggest a critical time for the effect of GTH-antiserum spanning, at most, the first 6 days after birth. It is tempting to speculate that the effect of the GTH-antiserum in these experiments may have been mediated by a temporary blockade of gonadotropin-dependent testicu-

6 ROLE OF GONADOTROPIN IN NEONATAL MALE RAT 449 lar hormone secretion. This hypothesis is particularly attractive when one considers that the antiserum had to be administered during the first 5 days of life, a period when androgen is required for masculinization of certain sex-related systems. Indeed, the concept of a critical time for the early action of testicular hormone is well-founded. astration of the neonatal male prevents both normal development of masculine sexual behavior patterns (Whalen and Edwards, 1967) and differentiation of the male-type hypothalamic control of gonadotropin secretion (Gorski and Barraclough, 1963). In order to obtain either of these effects, castration must be performed during roughly the first 5 days of life. Also, testicular concentrations of both steroidogenic enzymes (Niemi and Ikonem, 1963) and testosterone (Resko et al., 1968) as well as plasma levels of testosterone are relatively high during the first few days after birth and then decline until near the time of puberty. The brain may be highly sensitive to androgen during very early life since the uptake of labeled testosterone by the hypothalamus is greater on days 1, 2, 3, and 5 than on days 10 and 20 of life (Diamond and Dale, 1967). In our experiments testosterone propionate on day 1 was able to partially prevent the sterilizing effect of GTH-antiserum on days 1-5. This observation supports the view that the antiserum effect is mediated by a block of gonadotropin-dependent steroidogenesis. The lack of complete reversal of the antiserum effect may have been related to the fact that the testosterone was administered only on day 1 while the antiserum was given on days 1, 3, and 5. HG on day 1 may have been only partially effective for the same reason. Our studies do not directly indicate the point of fertility breakdown in the adult, sterile males. However, a priori one might list a number of possible explanations for male sterility. Foremost among these would be inadequate sperm or seminal fluid or decreased sexual behavior. Any of these situations could develop as a result of insufficient testosterone secretion during adulthood or they might develop independently of testosterone levels. In the present experiments the testes of antiserum-treated, sterile males appeared histologically normal at adulthood. Thus, it seemed unlikely that sterility was due to insufficient spermatic development. However, the possibility of inadequate secretion of seminal fluid cannot be ruled out. It also appeared unlikely that poor androgen production could have been the sole basis for sterility since males which had received GTH-antiserum on days 1-5 and saline on day 1 were completely refractory to testosterone priming during adulthood (Fig. 3). In contrast, animals which had received antiserum on days 1-5 Males Treated With AntIserum Days 1,3,5 Male. Treated With Antiserum Days 1, 3.5 and HG Day I Males Treated With MRS Days 1,3.5 I di E 0 01 U Before Priming After Priming Before Priming Alter Priming 5:17 8:19 I [] Pseudopregnant (P.) #{149} Pregnant (P) FIG. 3. Results obtained after testosterone propionate treatment during adulthood in two types of experimental male rats. Results obtained with the same males before testosterone priming are reproduced here for comparison, as are results obtained with NRS-treated males. 518 I

7 450 GOLDMAN AND MAHESH and HG on day 1 responded to testosterone priming by displaying normal fertility. It may be that the latter males were only relatively insensitive to androgen and that this insensitivity was overcome by the large dosage of exogenous steroid. Thus, it seems that an intermediate grade of sterility may be produced, probably depending upon the degree of gonadotropic stimulation and/or androgen available during the first few days of neonatal life. An analysis of the data provides some direct support for the hypothesis of inadequate sexual behavior by the sterile males. This can be seen in the observation that even when the percentages of pregnancies and pseudopregnancies produced by these males are summed the result is only about one-half as great as the percentage of pregnancies induced by NRS-treated males. The suggestion is that the sterile males were relatively inefficient in providing cervical stimulation since female rats become pseudopregnant following copulation even when ejaculation fails to occur (Everett, 1966). In behavioral tests male rats generally show higher scores after gaining experience (Rosenblatt, 1965). It is unlikely that experience was a factor in the present experiments, however, since no increase in fertility was apparent in successive tests with individual males. This was true for both sterile and normal animals. In conclusion, we suggest that GTH-antiserum on days 1-5 may block gonadotropin stimulation of the testes at a time when testicular secretion is normally involved in the development of systems which ultimately control male sexual behavior. In fact, male rats which are castrated within a few days after birth appear to be incapable of displaying a complete ejaculatory response even following testosterone priming during adulthood (Hendricks, 1969). Thus, if GTH-antiserum treatment does indeed inhibit steroidogenesis in the neonatal male, it seems reasonable to expect that sexual behavior might be impaired, resulting in sterility. In view of the arguments presented above, it is perhaps surprising that antiserum treatment on days 1-5 failed to prevent development of the masculine-type hypothalamic control of gonadotropin release since masculinization of this control mechanism is dependent upon secretion of testicular hormone(s) during the first few days of life. However, it may be that less androgen is required to masculinize the hypothalamic control mechanism for gonadotropin release than is required for establishment of normal male fertility. In the present experiments the necessary androgen for masculinization of the control of gonadotropin release may have been derived from nongonadotropin-dependent testicular secretion. Alternatively, the antiserum treatment may have failed to totally block endogenous gonadotropic activity. There is precedent for the concept of independent steroidal regulation of development (during neonatal life) of the gonadotropin release mechanism and of the mechanisms which control sexual behavior, respectively. For example, female rats which were treated with 10 sg testosterone propionate on day 2 of life became anovulatory; however, such animals showed normal female-type sexual behavior when primed with estrogen and progesterone during adulthood (lemens and Gorski, 1968). Also, female rats which were treated with deoxycorticosterone acetate during the first 5 postpartum days showed deficits in sexual behavior but displayed normal ovulatory cycles (Thomas and Gerall, 1969). The present experiments do not distinguish between the effects of endogenous LH and FSH, respectively, in neonatal rats. The results of Lostroh (1969) suggest that in juvenile male rats both hormones are required for normal maturation of spermatogenic function. However, to our knowledge there is no information concerning the respective functions of

8 ROLE OF GONADOTROPIN IN NEONATAL MALE RAT 451 these hormones in the neonate. Highly specific FSH- and LH-antisera would be of use in this regard. AKNOWLEDGMENTS The authors thank the Endocrine Study Section, National Institutes of Health for the gift of ovine gonadotropins used in these studies. REFERENES LEMENS, L. G., AND GORSKI, R. A. (1968). Induclion and facilitation of female mating behavior in rats treated neonatally with low doses of testosterone propionate. Endocrinology 84, DIAMOND, M., AND DALE, E. (1967). Distribution of radiolabelled steroid after administration to the neonatal rat. Anat. Rec. 157, 234. EVERETT, J. W. (1966). The control of the secretion of prolactin. In The Pituitary Gland (G. W. Harris and B. T. Donovan, eds.), Vol. II, pp University of alifornia Press, Berkeley and Los Angeles. GOLDMAN, B. D., GRAZIA, V. R., KAMBERI, I. A., AND PORTER, J.. (1970). Serum gonadotropin levels in neonatal rats. Proceedings of the 52nd Meeting of the Endocrine Society. GOLDMAN, B. D., AND MAHESH, V. B. (1968). A possible role of acute FSH-release in ovulation in the hamster, as demonstrated by utilization of antibodies to LH and FSH. Endocrinology 84, GORSKI, R. A., AND BARRALOUGH,. A. (1963). Effect of low doses of androgen on the differentiation of hypothalamic regulatory control of ovulation in the rat. Endocrinology 73, HARRIS, G. W. (1964). Sex hormones, brain development and brain function. The Upjohn Lecture of the Endocrine Society, Endocrinology 75, HENDRIKS, S. E. (1969). Influence of neonatally administered hormones and early gonadectomy on rats sexual behavior. J. orn p. Physiol. Psychol. 69, JAcOB50HN, D. (1966). The techniques and effects of hypophysectomy, pituitary stalk section and pituitary transplantation in experimental animals. In The Pituitary Gland G. W. Harris and B. T. Donovan, eds.), Vol. II, pp University of alifornia Press, Berkeley and Los Angeles. LOsmoH, A. J. (1969). Regulation by FSH and ISH (LH) of reproductive function in the immature male rat. Endocrinology 85, NIEMI, N., AND IKONEM, K. (1963). Histochemistry of the Leydig cells in the postnatal prepubertal testis of the rat. Endocrinology 72, PARLOW, A. F. (1961). Bioassay of pituitary luteinizing hormone by depletion of ovarian ascorbic acid. In Human Pituitary Gonadotropins A. Albert, ed.), p harles Thomas, Springfield, Illinois. RESKO, J. S., FEDER, H. H., AND GOY, R. W. (1968). Androgen concentrations in plasma and testis of developing rats. J. En.docrinol. 40, ROSENBLATT, J. S. (1965). Effects of experience on sexual behavior in male cats. In Sex and Behavior (F. A. Beach, ed), pp Wiley, New York. STEELMAN, S. L., AND POHLEY, F. M. (1953). Assay of follicle-stimulating hormone based on the augmentation with human chorionic gonadotrophin. Endocrinology 53, TIIOMAS, T. R., AND GERALL, A. A. (1969). Dissociation of reproductive physiology and behavior induced by neonatal treatment with steroids. Endocrinology 85, WHALEN, R. E., AND EDWARDS, D. A. (1967). Hormonal determinants of the development of masculine and feminine behavior in male and female rats. Anat. Rec. 157,