EXPERIMENTAL AND MOLECULAR PATHOLOGY 51, 1-7 (1992)

Ancylostoma ceylanicum Infection in Female Hamsters: An Observation on Altered Reproductive Function’

SUMAN GUPTA, JANMEJAI K. SRIVASTAVA, BRIJESH MALAVIYA,~ AND JAGDISH C. KATIYAR~

Division of Parasitology and ‘Endocrinology, Central Drug Research Institute, Lucknow 226001, India

Received January 24, 1991, and in revised form March 18, 1992

Reproductive performances of female hamsters were investigated during Ancylostoma ceylanicum (hookworm) infection. Animals having the highest levels of infection (34.96 f 1.1 I worms) showed degenerative changes in the reproductive system. Ovaries of infected animals contained a few primary or secondary follicles. On cocaging with males of proven fertility, only 7-8% (80% in controls) of the infected females mated but did not conceive as evidenced by the absence of corpora lutea or implantation sites on day 10 postcoitum. Animals with low worm burdens (5.94 * 0.65 worms), however, showed almost normal fertility. The uterine weight bioassay and compensatory ovarian hypertrophy suggest strong suppression of pituitary gonadotrophin contents in infected females. Resorptive effects on the pregnancy outcome of infected female hamsters were also recorded. 8 1992 Academic

Press. Inc.

INTRODUCTION

Parasitic infections, due to their general debilitating effect on body systems, are known to affect various functional components. Ancylostomiasis is a major prob- lem in the tropics and subtropics and global endemicity has been estimated to be over 1000 million (Janssens, 1985). The disease causes severe anemia, gastroin- testinal disturbances, and ulcers, along with physical, mental, and sexual retar- dation (Watson, 1960; Tandon et al., 1969; Areekul, 1977; Sheehy et al., 1982; Loureiro ef al., 1983; Schofield, 1985). During pregnancy the infection may be associated with changes in renal function (Faust and Russel, 1964) culminating in toxemia and albuminuria.

From a physiological viewpoint all systems related to reproduction must work under optimal conditions and in harmony for any species to reproduce. In our previous report (Katiyar et al., 1989) we assessed the effects of Ancylostomu ceylunicum infection in male hamsters and severe derangement in the male gonads was noted. The effects included functional disturbances in spermatogenesis and in the hypophyseal-gonadal axis. Since any alteration in the hypophyseal-gonadal axis is also expected to be evident in females, the present study was undertaken to record the effects of A. ceylunicum infection on the reproductive performance of female hamsters.

MATERIALS AND METHODS

Experimental Animals

Laboratory-bred parasite-free hamsters (70-80 g) from the Institute’s animal house facility were used as the experimental host. Immature female mice used in

i CDRI Communication No. 4669. 3 To whom correspondence should be addressed.

1

0014-4800/92 $5.00 Copyright 6 1992 by Academic Press, Inc. AU rights of reproduction in any form reserved.

2 GUPTA ET AL.

the bioassay of pituitary gonadotrophins were also procured from the healthy inbred stock maintained in the Institute’s animal facility. The animals were housed in appropriate plastic cages and maintained under controlled climatic con- ditions, 25 +- 2°C and 40-50% humidity with a 12-hr light/dark photo period. They received standard rodent pellet (Lipton, India, Ltd.) and water ad Zibitum. Ani- mals were hemicastrated and laparatomized under light ether anesthesia; how- ever, deep ether anesthesia was administered before sacrifice.

Parasite Culture and Experimental Infections

A. ceylanicum infective larvae (Ls) were harvested from 8 to lo-day-old co- proculture (Gupta et al., 1987). Varying numbers of L, (10 L,-70 L3) in 0.2 ml of distilled water were administered orally to hamsters as per experimental design. Such inoculation has been shown to attain potency around Day 18 postinfection (p.i.) (Srivastava et al., 1986; Gupta et al., 1987).

Effect on Fertility and Ovulation

A total of 103 female hamsters infected with different inocula (10 Ls, 25 L,, 50 L,, and 70 L3) were cocaged with males of the same age with proven fertility in the ratio of 2: 1 and allowed to cohabitate for 12-15 days, covering a period equivalent to three estrous cycles. Simultaneous cocaging of 47 normal uninfected animals was also done. All females were checked daily for presence of sperm in the vaginal smear (Ward, 1946), and those showing sperm-positive smears were sep- arated and caged individually. On Day 10 postcoitum each female was laparat- omized and the numbers of corpora lutea and implantation sites were counted. Animals with normal implants were allowed to go to term and their litter size was recorded.

Those that did not mate during prolonged exposure to males were sacrificed and their ovaries were examined. The fallopian tubes and uteri were flushed with physiological saline in a hypodermic needle using a surgical microscope, and the number of ova released was counted (Graves, 1945).

Ovarian Histology

The ovaries of normal and infected hamsters having the highest level of infec- tion (3UO worms) were isolated and fixed in Bouin’s fluid for routine histological studies.

Effect on Pituitary Gonadotrophins

In view of cyclic variations in the total gonadotrophin content in the pituitaries of female hamsters, studies were conducted on males, which do not exhibit pul- satile release of gonadotrophins. Hypophyseal gonadotrophin content of pitui- taries obtained from infected (25-37 worms) and normal male hamsters was as- sayed using the mouse uterine weight bioassay (Klinefelter et al., 1943). Briefly, pituitaries from 60 infected and 60 normal male hamsters were collected, weighed, and stored in chilled acetone. The glands were homogenized in chilled distilled water. The extract was diluted with distilled water containing 1 mg of pituitary gland (intact weight) in a 0.2-ml volume. Female mice, age around 21 days, were used in three groups of eight each. Mice of groups I and II received six subcuta- neous injections (0.2 ml) of normal and infected pituitary extract as described by Klinefelter et al. (1943), and group III received only normal saline as a sham

FEMALE REPRODUCTION DURING ANCYLOSTOMIASIS 3

treatment. Animals were sacrificed 24 hr after the last injection. The vaginal opening was checked and uteri were dissected out rapidly, cleaned, blotted, and weighed.

Compensatory Ovarian Hypertrophy

In an attempt to monitor the effect of A. ceylanicum infection on pituitary gonadotrophins in vivo, the compensatory ovarian hypertrophy assay was em- ployed, in which the increase in weight of the remaining ovary after removal of one ovary is recorded. The increase is caused by an increase in gonadotrophin secretions resulting from lower estrogen levels (Greenwald, 1960; Edgreen et al., 1965; Brinkley and Young, 1969). Infected female hamsters harboring 15- to 16- day-old infections were unilaterally ovariectomized. A similar number of unin- fected hamsters were also operated upon. Isolated ovaries were weighed and the number of corpora lutea was counted.

On Day 14 postovariectomy the animals were sacrificed, the remaining ovary was excised and weighed, and the corpora lutea were counted. Individual worm burden was also registered.

Effect on Pregnancy Outcome

A number of normal female hamsters were cocaged with normal males. Mating was confirmed by examination of vaginal smears (Ward, 1946). Mated hamsters (40) were divided into two groups, a and b. Each was further divided into two subgroups a, and a2 and b, and b2. Animals of group a, (5 hamsters) were kept as uninfected control; however, group a2 (15 hamsters) was fed orally with 50 adult worms on Day 2 postcoitum (p.c.) to study the effects on early events of preg- nancy. Similarly, group b, (5 hamsters) was kept as uninfected control and infec- tion was administered to 15 hamsters (b2) on Day 10 p.c. to monitor abortifacient effects.

All hamsters were allowed to go to term and litter size was individually re- corded. Those that did not deliver were autopsied after the expected date of delivery to record the gynecological outcome.

Statistical Analysis

Data were expressed as the mean ? SE. A statistical analysis was carried out by unpaired Student’s t test and Fisher Behran’s d test.

RESULTS

Impact on Fertility and Ovulation

The infected females showed an indirect correlation between worm burden and reproductive performance (Table I). There was a substantially lower mating rate in infected hamsters with relatively fewer corpora lutea, implantation sites, and litter size. In contrast, in the majority of the normal mated hamsters the pregnancy was established. They had many corpora lutea and implantation sites. Further, these animals had larger litter sizes.

The washings of fallopian tubes and uteri of 10 infected hamsters (harboring an average of 24 worms) autopsied at the estrous stage revealed that in the majority of hamsters (80%) the ova were absent. In two animals, however, one ovum in

4 GUPTA ET AL.

‘TABLE I Reproductive Performance of Female Hamsters during A. ceylunicum Infection

Worm load Group (mean 2 SE)

Control Nil

Infected I 5.94 2 0.65

II 15.76 f 0.87 III 26.23 k 0.54 IV 34.96 +- 1.11

No. of animals

(replicates) matedkocaged

38147 (9)

12/17 (4) 5113 (4) 5/21 (6) 4152 (12)

Percentage of mating

rate

80.85 36 (11.40 f 0.46)

70.58 38.46 41.66 07.69

Female became pregnant (No. of

corpora lutea, mean f SE)

Implantations Litter size (mean f SE) (mean 2 SE)

9.5 + 0.31 5.34 2 0.36

9 (5.38 k 0.75) 3.93 -t 0.85 2.73 f 0.67 1.0 (6.00 k 00) 6.00 + 00 Nil 1.0 (0.57 + 0.39) 6.00 ” 00 Nil

Nil Nil Nil P < 0.001* Pi 0.001* P < 0.001*

* Normal vs infected.

each was observed. Uninfected normal females released 5-l 1 ova and the ovarian appearance was normal (not shown in Table I).

Effect on Pituitary Gonadotrophins

Mouse uterine weight bioassay revealed that in infected male hamsters (25-38 worms), there was a depletion in the gonadotrophin level. Mice receiving pituitary homogenate from normal hamsters showed a significant gain (P < 0.001) in uterine weight (Table II), and their vaginas were open. Contrary to this, mice adminis- tered infected pituitary homogenate showed low weight gain and their vaginal openings were closed.

Compensatory Ovarian Hypertrophy

In unilateral ovariectomy the ovaries of infected females (15.9 + 2.02 worms) revealed only a 15.38% weight gain (hypertrophy) and comparatively no increase in the number of corpora lutea (1.40 k 0.40 vs 1.80 rt 0.81). In contrast, the ovaries of similarly operated normal females showed 97.13% hypertrophy with a significant increase in the number of corpora lutea (4.42 + 0.32 vs 10.25 + 0.49) (Table III).

Effect on Pregnancy Outcome

The lower establishment of adult worms from the inocula given either on Day 2 (5-8 worms) or on Day 10 p.c. (7-14 worms) did not change the gestational

TABLE II. Effect of A. ceylunicum Infection on Pituitary Gonodotrophin Contents of Male Hamsters

No. of mice Uterine weight (mg) Group (replicates) (mean f SE)

I. Normal control (received pituitary homogenate from normal hamsters) 8 (2) 33.13 2 3.06*

II. Experimental (received pituitary homogenate from infected hamsters) 8 (2) 14.38 -r- 1.446

III. Control (received NS only) 8 (2) 6.00 f 0.64”

Note. Statistical significance: P < 0.001 for groups c vs a, c vs b, and 0 vs b.

Vaginal opening

Open

Closed Closed

FEMALE REPRODUCTION DURING ANCYLOSTOMIASIS 5

TABLE III Effect of A. ceylanicum Infection on the Action of Gonadotrophin Contents of Unilaterally

Ovariectomized Hamsters

On castration, day 15 p.i. On autopsy

Weight of Weight of Percentage No. of isolated remaining change in animals Worm load ovary (ms) No. of C.L. ovary (mg) No. of C.L. OVtiiUl

Group (replicates) (mean 2 SE) (mean f SE) (mean f SE) (mean + SE) (mean k SE) weight

Control 12 (2) Nil 5.92 f 0.37” 4.42 + 0.32* 11.67 k 0.46a’ 10.25 f 0.496 +97.13 Infected 10 (2) 15.9 f 2.02 6.50 k 0.W 1.40 f 0.4od 7.50 + 0.61” 1.80 2 0.81d’ + 15.38

Nore. Statistical significance: a vs a’lb vs b’, P < 0.001; c vs c’ld vs d’, not significant.

pattern, except for the small litter size. However, the higher worm load affected the pregnancy outcome (Table IV). Hamsters infected on Day 2 p.c. and harbor- ing l&15 worms resulted in few living and partially resorbed implants. However, hamsters having 16-26 worms showed a complete absence of pregnancy. Simi- larly, infection on Day 10 p.c. (worm load, 22-27) resulted in either no implanta- tion sites or 4-7 resorbed fetuses.

DISCUSSION

A. ceylanicum is a common enteric infection causing a general debilitating effect on the infected population. However, little attention has been paid to the effects of infestation on reproductive functions. In our previous communication we reported inhibition of spermatogenesis along with atrophic changes in the secondary sex organs, viz., epididymis, prostate, and seminal vesicles. All of these pointed to alterations in the hypophyseal-gonadal axis (Katiyar et al., 1989). Results of the present studies revealed a linear relationship between worm load and degree of reproductive dysfunction in female hamsters. As is evident, A. ceylanicum infection inhibited ovarian function and arrested follicular develop- ment at the secondary phase. The follicles were devoid of antrum. Similar results were obtained with mated female hamsters where there was a distinct reduction in the number of corpora lutea. With heavy worm load, no ovulation was detected. These studies indicate an effect on the hypophyseal-gonadal axis. Bioassay for hypophyseal gonadotrophin contents revealed reduced gonadotrophins in infected hamsters; uterine weights of mice receiving extracts of pituitaries from infected hamsters did not show significant gains in comparison to those of mice receiving

TABLE IV Effect on A. ceylanicum Infection on Pregnancy Outcome of Hamsters

Group

No. of Day of Worm mated infection recovery

females postcoitum on autopsy

a, Control 5 a, Infected 5

5 5

b, Control 5 b, Infected 5

10

- 2 2 2

- - 5-12 babies born 10 7-14 3-5 babies born 10 22-27 Either no implant or 4-7 resorbed fetuses

5-g lo-15 16-26

Observations (average pregnancy outcome)

5-12 babies born 3-6 babies born 4 resorbed and 3 living foetuses Either no implant or I-10 resorbed implants

6 GUPTA ET AL.

pituitary extracts from normal hamsters. Vaginal openings of these mice were also closed, suggesting decreased gonadotrophin levels in the pituitaries of infected hamsters. In view of the pulsatile release of gonadotrophin in females, male ham- sters were used for these studies because they show a constant and steady syn- thesis and release of gonadotrophins. In an attempt to further elaborate on this point, compensatory ovarian hypertrophy following hemicastration of normal and infected hamsters was recorded. It is pertinent to note that on Day 15 pi. ovaries revealed degenerative changes and an increase in dry weight due to fibrosis. This pathology resulted in subdued ovulation and there was apparently no compensa- tory hypertrophy, as is often observed after hemicastration (Edgreen et al., 1965; Brinkley and Young, 1969) in normal animals. Since the corpora lutea are the largest and most persistent of ovarian elements, the weight increase as noted in the ovaries of uninfected hemicastrates probably results largely from ovulation and postovulatory luteinization in these animals. Inhibition of ovulation, as evi- denced by the absence of corpora lutea in the remaining ovaries of hemicastrated infected hamsters, pointed to changes in LH synthesis and release. However, involvement of FSH cannot be ruled out, since Gorski and Barraclough (1962) and Swanson and Vanderwerften Bosche (1964) have shown ovarian compensatory hypertrophy in anovulatory, androgen-sterilized rats which appear to be largely FSH dominated. Inhibition of ovarian weight gain in infected hamsters thus shows the effect on both gonadotrophin components. The effect of FSH is further indi- cated by the absence of a vaginal opening in mice receiving pituitary homogenates from infected hamsters. The mechanisms involved in alterations in hypophyseal function are not clear and need further investigation.

ACKNOWLEDGMENT One of authors (J. K. S.) is grateful to CSIR, New Delhi, for financial assistance in the form of

Research Associateship.

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