Ovarian hypoplasia in heifers due to germ cell weakness

ELSEVIER

OVARIAN HYPOPLASIA IN HEIFERS DUE TO GERM CELL WEAKNESS I. Settergren Department of Obstetrics and Gynaecology Faculty of Veterinary Medicine, SLU Uppsala, Sweden Received for publication: Accepted:

April 12,

1996

September

26,

1996

ABSTRACT Ovarian hypoplasia due to germ cell weakness was found in 18 heifers of the Swedish Red and White breed simultaneously with a sharp increase of the same syndrome in bulls of the same breed. The heifers were examined because they had not shown estrus or had not become pregnant after several months on pasture together with a fertile bull. After palpation of the ovaries per rectum they were classified as normal, partially hypoplastic or totally hypoplastic. The heifers were slaughtered and the numbers of primordial follicles were counted in the serially sectioned ovaries. The average numbers of follicles per ovary in the 3 diagnostic groups were 187 16, 6617 and 4761, respectively. The numbers of primordial follicles in hypoplastic ovaries were significantly lower than in normal ovaries but were still within normal limits for heifers. However, the hypoplastic ovaries were very small, and the totally hypoplastic ones weighed only 1 g or less. The growth of the primordial follicles was disturbed and very few developed into Graafian follicles. There were also many empty cortical cords and anovular follicles. The heifers had all been sired by bulls belonging to breeding lines where germ cell weakness had occurred. 0 1997 by Elsevier Science Inc. Key words: heifers, ovaries, hypoplasia, anestrus, hereditary

Acknowledgements: The author thanks Dr. K.-A. Ahlin for statistical analysis of the data, Ms. Amrika Rikberg for excellent assistance in the laboratory, and Ms. Birgitta Berner for preparation of the manuscript.

Theriogenology 47531-539. 0 1997 by Elsevier Science

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532 INTRODUCTION

Ovarian hypoplasia is a congenital, mostly hereditary condition which can be unilateral or bilateral and total or partial. The affected ovaries are small and usually contain a low number of primordial follicles or no follicles at all. Further, the quality of the primordial follicles is lower than normal so they do not develop normally. Ovarian hypoplasia has been extensively studied in the Swedish Highland Breed (SKB; 4, 5, 6, 7, 11). It was shown that the hypoplastic ovaries contained no follicles in the whole ovary or part of it. The hypoplasia was usually leftsided (- 88 %) but could also be rightsided ( - 4 %) or bilateral ( - 8 %). Genetic analysis has revealed that the hypoplasia is caused by a recessive gene with incomplete penetrance (3). In the Swedish Red and White Breed (SRB), a few cases of testicular hypoplasia were reported by Lagerliif (4). Some 20 years ago there was a sharp increase in the frequency of testicular hypoplasia in Swedish Red and White Breed bulls sired by 2 half-brothers and their sire. The hypoplasia was principally different from that found in Swedish Highland Breed bulls, and has been called germ cell weakness (12). A few cases of a similar type of gonadal hypoplasia had been found over the years in Swedish Red and White Breed heifers, but some 20 years ago there was an increase simultaneously with the increase of germ cell weakness in bulls. A preliminary report on clinical hypoplasia of the ovaries in Swedish Red and White Breed heifers bred by a sire with a high frequency of germ cell weakness in his sons has been published (10). In 110 daughters the frequency of partial ovarian hypoplasia was about 30%. The aims of the present investigation were to study the histology and follicular system of the ovaries of Swedish Red and White Breed heifers with clinically diagnosed hypoplasia. MATERIALS AND METHODS The experimental animals consisted of 18 Swedish Red and White Breed heifers. The heifers were examined by field veterinarians at 2 or 3 AI associations because they had not shown any symptoms of estrus or had not become pregnant after 4 to 6 mo on pasture with a bull. The reproductive tract was examined by palpation per rectum and the ovaries were classified as normal, partially hypoplastic or totally hypoplastic, as shown in Table 1. The heifers were about 2 yr old, with a range of 18 to 32 mo. They were all sired by bulls belonging to breeding lines in which testicular hypoplasia due to germ cell weakness had occurred (12). The heifers were slaughtered and the ovaries were examined macroscopically and subsequently fixed in Bouin’s solution. After 24 h the ovaries were transferred to 70% alcohol and sent to the Veterinary College where they were further processed and embedded in histowax. The ovaries were serially cut in 10 p thick sections. Every 20th

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section was kept and stained with hematoxylin-eosin. Pieces were also taken from some uteri in heifers with bilateral total ovarian hypohlasia and processed as reported above. The number of primordial follicles were counted in every 100th section of the ovaries, with the oocyte nucleus as the marker. When there were very few primordial follicles every 20th section was examined. Because an oocyte nucleus can appear in more than one of the 10-p thick sections, correction was made in the calculation of the total number of primordial follicles according to a method described elsewhere (1). According to the formula the number of primordial follicles counted should be multipled by the factor 0.43 to get the correct number. Statistical analysis was performed by a two-sample t-test with the program Minitab for Windows (release 10.2). RESULTS In the 9 heifers with clinical bilateral ovarian hypoplasia (H+H in Table l), the ovaries were very small, weighing 1 g or less. The surface of the ovaries was wrinkled and without signs of corpora lutea (CL) or Graafian follicles. None of the heifers in this group had shown any signs of es&us. Histological examination of the totally hypoplastic ovaries showed a cortex with a varying number of primordial follicles (Figure 1A) from a minimum of 48 to a maximum of 13463, as shown in Table 1. However, the primordial follicles did not develop normally but underwent atresia either directly or after some growth. A few grew to small Graafian follicles and then became atretic, often with luteinization of the theta intema. Besides the primordial follicles there were many anovular cortical cords and follicles (Figure 1B). They seemed to be formed from cortical cords in the absense of oocytes. The anovular follicles could develop further like growing follicles (Figure 1B arrow) but would never form an antrum. The uteri of heifers with bilateral total ovarian hypoplasia were very small, weighing less than 100 g, and with a soft consistency. Histological examination showed a prepubertal appearance. The ephitelium of the endometrium as well as in the endometrial glands was very low, and there were either no lumina or very small lumina in the glands (Figure 2B). The myometrium was also very thin and had tightly packed nuclei. The partially hypoplastic ovaries weighed about 2 to 4 g and contained 0 to 14889 primordial follicles, with an average number of 6617 per ovary. This was more than the average of 4761 in the totally hypoplastic ovaries but the difference was not statistically significant. The 2 heifers with bilateral partial ovarian hypoplasia had quite different

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Table 1.

Number of primordial follicles in the ovaries of heifers in 3 different diagnostic groups

Identity of heifers

Clinical diagnosis Left Right ovary ovary

2379

2416 2175 3696 3189 2961 3336 1103 2264

Number of urimordial follicles Left Right ovary ovary

H H H H H H H H H Average

2480 2481 3106 2482

H H H P

P P P H Average

2483 3697

P P

P P Average

2279 2350 2498

P N P

N P N Average

H= totally hypoplastic

P= partially hypoplastic

10110 9503 5561 4470 5556 3810 3870 918 346

13463 7317 5404 5891 4033 5478 3973 1224 48

4905

5203

3251

6291 3178 418

11576 1653 4147 1052

3265

4607

14889 321

13548 0

7605

6774

11733 19479 4543

34000 9959 2670

11918

15543

N= normal.

numbers of primordial follicles in the ovaries. Heifer 3697, which was the oldest of the heifers (32 mo), had only 321 follicles while Heifer 2483 had 28437 follicles. The

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Figure 1

535

A) One normal primordial follicle and some anovular cortical cords in a totally hypoplastic ovary. Hematoxylin-eosin (x 200) B) Anovular cortical cords in a row and a growing anovular follicle (arrow) in a totally hypoplastic ovary. Hematoxylin-eosin (x 80)

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first heifer had a few Graafian follicles in each ovary and remnants of what most probably had been luteinized follicles, while the latter heifer had a few Graafian follicles in each ovary but no signs of CL or luteinized follicles. No estrus had been detected in the 2 heifers. The 3 clinically normal ovaries combined with partially hypoplastic ovaries weighed 4.7 to 8.2 g. The average number of primordial follicles in the normal ovaries was 18716, which was significantly more than the number in partially (P=O.O45) and totally hypoplastic ovaries (P =O.OOOS). In Heifer 2279 there were 3 CL of apparently the same age in the right ovary. The left ovary, where the medial part was very thin and wrinkled, had a 16-mm follicle with cystic atresia in the lateral part. Heifer 2350, an 18 mo old, had 16 Graafian follicles 2_ 1 mm in the normal left ovary, of which 13 were atretic and 3 were norm.al. In the right partially hypoplastic ovary there were 7 follicles 2. 1 mm, of which 4 were atretic and 3 were normal. Heifer 2498 had a 16-mm CL in the right ovary (normal) and remnants of an earlier corpus luteum. In the left partially hypoplastic ovary there were 3 normal follicles and 1 atretic follicle 2 1 mm. DISCUSSION The 9 Swedish Red and White breed heifers with total bilateral ovarian hypoplasia in our study had an average of approximately 10,000 primordial follicles in the ovaries in contrast with that of the Swedish Highland breed, in which 10 heifers with total bilateral ovarian hypoplasia had no follicles at all (11). This shows that there is a basic difference in the gonadal hypoplasia between the 2 breeds. In the defective Swedish Red and White Breed heifers the number of follicles is almost the same as in normal animals, but the follicles do not develop normally and undergo atresia at an early stage. Very few of them develop into Graafian follicles before they become atretic, often with some luteinization of the theta interna. The atresia in Graafian follicles often seemed to start in the oocyte, which is not common in normal ovaries (Figure 2A), indicating that this type of ovarian hypoplasia is caused by a weakness in the oocytes corresponding to germ cell weakness in bulls of the same breed (12). Ovarian hypoplasia in Swedish Red and White Breed heifers nearly always occurs bilaterally, which is also different from that in Swedish Highland Breed heifers, in which one of the ovaries can be totally hypoplastic while the other remains clinically normal and thus the heifer can maintain normal fertility. Germ cell weakness has not been described in any other cattle breed. However, in Romney ewes a similar condition was reported (2). Ewes heterozygous for the Inverdale gene had an increased ovulation rate, while homozygous ewes had bilateral gonadal hypoplasia with streak gonads, with a volume of about l/8 of normal ovaries. Although these affected ovaries had a large number of primordial follicles normal

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Figure 2

537

A) Graafian follicle with the start of atresia in the oocyte in a partially hypoplastic ovary. Hematoxylin-eosin (x 200). B) Uterus of a heifer with bilateral total ovarian hypoplasia. Hematoxylin-eosin (x 80)

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follicular growth seemed to be blocked, and the other organs of the genital tract resembled those of prepubertal ewes. A condition similar to germ cell weakness has also been described in mice homozygous for the recessive gene for nudeness (8). These mice do not develop a thymus gland and the females show severe deficiencies in reproductive functions. At 1 mo of age no significant difference was found between the nudes and their normal littermates in the number of small or growing follicles, but by 2 mo the number of both small and growing follicles was lower in the nude mice, and at 4 mo ovulation had ceased. This rapid reduction in the number follicles resembles the situation in heifers with germ cell weakness. In nude mice, treatment with gonadotrophins restored the growth of follicles to normal values (9). Thus far, treatment with gonadotrophins of heifers with hypoplasia due to germ cell weakness has not been tested. As in bulls, the germ cell weakness syndrome in heifers most probably has a hereditary basis. All the heifers in our present study were sired by bulls from breeding lines where germ cell weakness has occurred. However, the study of the hereditary character of the syndrome has yet to be undertaken. REFERENCES 1. 2.

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Lintem-Moore S, Pantelounis EM. Ovarian development in athymic nude mice III. The effect of PMSG and oestradiol upon the size and composition of the ovarian follicle population. Mech Ageing Dev 1976;5:33-38. 10. Lundgren B. A preliminary report on hypoplasia of the gonads in the Swedish Red and White cattle (SRB) . Proc 7th Int Congr Anim Reprod 1972;3 : 1958- 196 1. 11. Settergren I. The ovarian morphology in clinical bovine gonadal hypoplasia with some aspects of its endocrine relations. Acta Vet Stand 1964;5 (Suppl 1): 108~~. 12. Settergren I, McEntee K. Germ cell weakness as a cause of testicular hypoplasia in bulls. Acta Vet Stand 1992;33:273-282. 9.