Morphological and Hormonal Features of an Ovine and a Caprine Intersex

W.T.K. Bosu and P.K. Basrur*

ABSTRACT Mots cles: intersexue, testosterone, plasma steroid concentrations in these chimeres sanguines, free-martins animals in relation to their cytogenetic A caprine and an ovine intersex ovins, free-martins caprins. make up and histological modifica- were examined to compare their mor- tions of the reproductive system. phological and hormonal features in light of their cytogenetic make-up. INTRODUCTION Both animals, registered as females at MATERIALS AND METHODS birth, developed male-like appearance Intersexuality, a well recognized and behaviour as they approached the condition in dairy goats is an impor- INTERSEX GOAT age of sexual maturity. Plasma testos- tant cause of caprine infertility. The A polled Toggenburg kid, approxi- terone concentrations in the intersexes recorded incidence of intersexuality in mately three months of age was pres- were similar to those in adult males of dairy goats ranges from 2 to 15% ented to the Ontario Veterinary Col- the respective species. Cytogenetic (1,2,3). The condition also occurs in lege on November 28, 1979 for analyses showed male and female cells sheep, although the incidence varies reproductive system evaluation. The in the blood while cultures of solid between breeds (4). Several investiga- kid was reported to have been born tissue contained only female cells sug- tors have described the anatomy, his- cotwin to a male kid and was regis- gesting that both were blood chimeras tology and cytogenetics of intersex tered as a female at birth. The dam was similar to the bovine freemartins. goats (3,5,6,7). On the other hand, polled but the sire was horned. The sire ovine intersexes are less well docu- exhibited unilateral testicular hypo- Key words: Intersex, testosterone, mented (8,9, 10). Data on steroid secre- plasia, the left testis being a third blood chimeras, ovine freemartins, tion in intersex sheep and goat are smaller than the right. caprine freemartins. scanty although Short et al (7) and The kid had the external appearance Hammerton et al (3) have studied the of a female. On clinical examination, testosterone, androstenedione and the most striking finding was the small RESUME progesterone concentrations in the vulva and the prominent clitoris (1 cm gonadal tissues and peripheral plasma long and 0.5 cm wide) protruding Cet article rapporte rexamen de deux of a few intersex goats and Saba et al from the vulva. Mammary gland intersexues, un caprin et un ovin, qui (10) have described the plasma testoste- development was within the normal visait a comparer leurs caracteristiques rone concentrations in a masculinized range for a doeling of corresponding morphologiques et hormonales, a la freemartin ewe. age. There were no structures palpable lumiere de leur constitution cytogene- Intersex goat or sheep represents a subcutaneously in the inguinal region. tique. Ces deux animaux, enregistres loss to the producer since they are gen- The kid was purchased for further comme femelles, a la naissance, deve- erally reared to maturity and then observation, hormonal studies and lopperent une apparence et un compor- found worthless for breeding pur- karyotype analysis. tement de male, a mesure qu'ils appro- poses. Of clinical interest is the unsub- The animal was housed in the same chaient de la maturite sexuelle. La stantiated, but commonly reported, pen with a three month old doeling teneur de leur plasma en testosterone se cyclical male-like and female-like until January, 1980 when the behav- revela comparable a celle des beliers et behaviour exhibited by some of these ioural and hormonal studies com- des boucs. Des analyses cytogenetiques animals. If true, such a behavioural menced. At weekly intervals from revelerent la presence de cellules males pattern would imply an alternating January 10, 1980 to the end of March, et femelles dans leur sang, tandis que les pattern of gonadal steroid secretion. the animal was observed for periods of cultures de muscles squelettiques ne The aims of the present study 30 minutes to determine its sexual contenaient que des celiules femelles, include determination of the sexual behaviour towards the pen mate. In indice que les deux intersexues corres- behaviour patterns of an ovine and a addition, it was exposed to an estrous pondaient a des chimeres sanguines, caprine intersex and the assessment of doe, or an estrogen treated doe, and a semblables aux free-martins bovins. the temporal changes in peripheral buck of identical age, for periods of

*Department of Clinical Studies (Bosu) and Department of Biomedical Sciences (Basrur), Ontario Veterinary College, University of Guelph, Guelph, Ontario NIG 2W1. Submitted August 18, 1983.

402 Can J Comp Med 1984; 48: 402-409. 5-20 minutes. A similar examination penned near the twins for comparative the other, but included interest in was carried out daily from April I behavioural and hormonal studies dur- estrous (or estrogen treated) does evi- (towards the end of breeding season) ing the period of one month. The sex- denced by perineal nosing, foreleg and between September 18 and 22 ual behaviour of the intersex towards striking and attempts to mount. It (onset of the next breeding season). its sibling (ram) and the ewe were never achieved a mount during any of Furthermore, the animal attendants recorded daily. Blood samples were the examination periods. However, it were instructed to record any sexual collected daily from the jugular vein of was observed mounting its pen mate behaviour pattern noted on days other the ram, the intersex and the ewe and and showing tongue lapping on other than the examination days. Sexual the plasma were stored at -20°C until occasions. The intersex showed mild behaviour was recorded as male-like if needed for assay. aggression towards the normal buck the kid showed interest in the estrous The intersex sheep was euthanized and on two occasions tried to butt him. doe, attempted to mount or mounted after a month of observation and the The intersex sheep maintained or showed hostile reaction to the buck. reproductive tract was dissected out male-like appearance and behaviour The behaviour was recorded as and examined. Representative sam- during the month of study. It dis- female-like ifthe buck showed interest ples from the reproductive tract were played aggressive behaviour which in the animal or if the animal was fixed and processed for histological was more noticeable when the ewe was receptive to the buck. examination. in estrus. On several such occasions Blood samples were collected via Testosterone and progesterone con- the intersex attempted to ram the jugular vein puncture from the inter- centrations in the plasma samples of animal attendant. sex, the pen mate (female) and an age the goats and sheep were determined The peripheral plasma testosterone matched buck, for steroid assay, at using radioimmunoassay methods and progesterone concentrations in weekly intervals from January to the (I 1,12). The validation of the method the intersex goat, a peer buck and a end of March and on three consecutive for goat plasma has been described doe are presented in Table I. The days each in April and September. The (12). Small samples of skeletal muscle period of study covered the latter part blood samples, drawn prior to behav- tissue were collected in phosphate buf- of the breeding season (January to ioural examination, were centrifuged fered saline (PBS) for explant culture April) and the onset of the next breed- and the plasma was collected and and chromosome analysis (5). ing season (September). The plasma stored at -20°C until needed for assay. progesterone concentrations in the The animal was euthanized on Sep- doe showed increase and decrease tember 22, 1980 and the reproductive RESULTS reflective of the normal patterns tract was examined. Representative expected during the estrous cycle. In pieces were fixed in Bouin's solution, The Toggenburg intersex goat grew both the buck and the intersex, the processed for 6 mu paraffin sections rapidly and acquired a masculine plasma progesterone concentrations and stained in haematoxylin and eosin appearance by six months of age at were never more than I ng/ mL and for histological examination. which time a faint but characteristic there were no indications of cyclical male-like odour was detectable. From changes. INTERSEX SHEEP the age of six months until the end of Testosterone patterns in the intersex A set of ten month old Dorset cross the observation period its behaviour and buck were similar. However, the twin lambs was presented for exami- was mainly male-like. There was no buck displayed higher testosterone nation at the Ontario Veterinary Col- development of the mammary gland. concentrations in the breeding season lege on October 31, 1980. One of these The display of male-like behaviour (January and September) while in the displayed signs of an intersex. At the varied from one examination time to intersex, the concentrations remained time of examination, the intersex weighed 35 kg and had the appearance of a maturing ram. However, the TABLE I. Plasma Steroid Concentrations in a Caprine Intersex at Different Times of The Year external genitalia consisted of a small Progesterone -ng/ mL Testosterone -pg/ mL vulva, with a protruding knob-like cli- Date Female Buck Intersex Female Buck Intersex toris measuring 1.5 cm long and I cm Jan. 10 0.60 ND ND 50 ND ND wide. The udder was evident. On 17 0.40 0.40 0.60 10 3950 2610 initial examination, the inguinal Feb. 15 2.60 0.10 0.40 40 5850 3450 region revealed no palpable structures. 21 ND 0.30 0.50 10 680 4100 However, two weeks later when the 29 6.10 0.50 0.30 20 1050 5250 sheep was shorn, palpation of the Mar. 7 9.10 0.50 0.40 30 610 5680 inguinal region revealed two oblong 14 0.40 0.70 0.60 ND 610 140 21 0.40 0.60 0.60 10 670 1950 structures soft in consistency, antero- 31 ND 0.70 0.30 ND 1020 1410 lateral to the mammary gland on each Apr. 1 ND 0.50 0.20 ND 660 630 side. Heparinized blood was collected 2 ND 0.60 0.30 ND 800 2090 aseptically for karyotype analysis. 3 ND 0.40 0.20 ND 1150 1320 The twins were acquired for further Sept. 18 3.90 0.60 0.60 50 9300 660 observation. A ten month old Dorset 19 4.20 0.40 0.30 40 7350 710 ewe obtained from a breeder was 22 5.60 0.40 0.20 40 7650 430

403 relatively high throughout the obser- like in appearance althoulgh the right brane was relatively normal although vation period. gonad was smaller. The billateral tubu- no germ cells were detected in any The plasma progesterone and test- lar structure joined at the caudal tubules (Fig. 3c). The epididymis on osterone concentrations in the intersex region in the area reminisscent of the both sides were well differentiated sheep, its sibling (ram) and a peer ewe cervix, and led into a narroiw vestibule. (Fig. 3d). are shown in Fig. 1. As with the goats, The histological architecture of the The tissue collected from the imme- the progesterone concentrations in the left gonad of the int(ersex goat diate vicinity of the gonads contained intersex sheep and the ram were never resembled an ovotestis (FFig. 3) with coiled tube-like structures resembling more than I ng/mL. The normal ewe large cysts on the periphery. The cysts oviduct lined by nonciliated and non- exhibited estrus on November 3 and 4 were lined by a single layer of cells secreting cells which were either flat or and the progesterone concentrations which, in some areas, prcDjected into cuboidal. The fluid filled tubular increased gradually thereafter to luteal the "lumen" of the cysts. structures detected around the gonads phase levels. The diameter and appea.rance ofthe were uterine horns lined by inactive The plasma testosterone concentra- seminiferous tubules variied greatly, epithelium on the luminal side (Fig. tions in the ram fluctuated between 0.5 some ofthem resembling degenerating 4a). The "body of the uterus" noted at and 6.9 ng/mL but most of the time, follicles while the ot hers were the caudal junction of the horns, on the concentrations were between 0.4 unequivocally seminiferouIs tubular in the other hand, was fairly large (Fig. and 2.8 ng/mL. On the other hand, the architecture (Fig. 3a). T~he tubules 4b) with extensive submucosal glands. testosterone concentrations in the were demarcated by a thic-k basement The epithelium on the luminal side was intersex were higher than in the ram membrane and were devc)id of germ discontinuous and the subepithelial and were between 1.4 and 5.2 ng/mL cells or a distinct lumen. 1rhe intersti- region of the glands contained abun- throughout the period of study. tium was extensive with a majority of dant leukocytes (Fig. 4c). The peri- Figure 2 illustrates the characteris- cells resembling lymph()cytes and phery of the uterine body contained tics of the reproductive tract in the fibroblasts (Fig. 3b). The iright gonad glands similar to the seminal vesicles intersex goat. The gonads on both was testes-like in histologi(cal features, of normal bucks (Fig. 4d). sides, surrounded by a fluid filled tub- the seminiferous tubules were uniform The reproductive tract from the ular structure, were round and testes- in diameter and the basement mem- intersex sheep is shown in Fig. 5. The gonads were hypoplastic testes and the tract was abnormal but more male- like. The histological features of the 6 gonads resembled that of a male with the exception that the seminiferous E * - RAM tubules were narrow and free of germ o - c EWE cells (Fig. 6). The most striking feature 4 x - INTERSEX of this animal was the abundance of z Leydig cells which were enlarged. The 3 duct system was well developed and n as were sex O 2 male-like the accessory 0 glands. The epithelial lining of the I ducts showed hyperactivity and a ten- dency for desquamation. In some 0 parts ofthe epididymis, the lumen was filled with amorphous, eosinophilic 7 material. No histological evidence of Mullerian duct ramifications was 6 noted in any of the ducts examined. -J E The external genitalia, including clito- C" ris and the vulva were similar to that noted in the caprine intersex. Z 4 0 + KARYOTYPE ANALYSIS o- +/ Examination of blood cultures tn 0 showed that both intersexes carried XX/ XY cells in peripheral blood although the proportion of male cells were relatively low in both animals ~. (Table 1I). The proportion of male 0 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 221 22 23 24 OCTOBER NOVEMBER DAY cells in the blood samples of the intersex sheep was lower than that of the intersex goat. Cultured cells in the Fig. 1. Plasma steroid concentrations in the intersex sheep, its ram sibling and an age-matched ewe. muscle biopsies of both intersexes were of XX constitution (Table II). 404 Davis (1) long before a chromosomal basis for the cattle freemartin was pro- posed (13). More recently, a few cases of freemartinism in goats and sheep were reported (8,14,15). Although the incidence of this condition is generally considered far lower than that in cattle, the reasons for the infrequent occurrence of freemartinism in females of heterosexual twin sets of goats and sheep is not known. It is possible that in these species fetal membrane fusion generally occurs late in gestation when hormonal exchange or cell migration from the male cotwin, has no masculinizing effect on the gonads of the female cotwin. It is also possible that the freemartin condition is not recognized as such since a definitive diagnosis of freemartinism requires karyotype examination. Thus a good proportion of ovine and caprine freemartins may be missed and grouped under "hermaphrodites". An interesting aspect of the present study is that although both animals were diagnosed as freemartins, they exhibited great variation in gonadal architecture, extent ofmasculinization of the duct system and accessory sex glands and pattern of sexual behavioural. Both intersexes displayed male L3Toburw% sexual behaviour but the sheep inter- Fig. 2. Diagram ofthe gross appearance ofthe genital tract ofthe intersex goat. 1: gonads, 2: uterine sex was more aggressive. In both horns, 3: body of the uterus, 4: enlarged clitoris. animals there was an indication of fluctuation in the male sex drive which appeared to peak in the presence of an TABLE II. Sex Chromosome Make-up of Metaphase Plates Analysed From Cultures ofa Caprine estrous female and during the breeding and an Ovine Intersex season. At no time did any ofthe inter- No. of Metaphases Percentage of sexes display female-like behaviour, Animal Tissue Examined XX XY XX Cells i.e. attracting the male or allowing the Goat Blood 54 45 9 16.6 male to mount. Muscle 26 58 0 0 The plasma steroid patterns shed Sheep Blood 63 58 5 7.9 light on the type of gonadal tissue Muscle 19 19 0 0 present in the intersex and the behavioural patterns observed. In the pres- these animals are genetic females sim- ent study, the testosterone concentra- DISCUSSION ilar to the freemartins in cattle (13). tions were similar or higher in the Both of these intersexes were born intersexes compared to peer males. Clinical diagnosis of intersex condi- twin to males and, as in cattle, were These findings are in accordance with tion in sheep or goats is usually based registered as females. Freemartinism is the results of an earlier study on inter- on gross observation of the external not frequently reported in goats and sex goats (7) and indicate the steroido- genitalia. Diagnosis can be confirmed sheep in which twinning is noted as genic competency of the gonads of at laparotomy on the basis ofthe gross often as single births. The apparent these intersexes. Fluctuations were not appearance of the gonads and the duct lack of freemartinism in goats and detected in the secretory patterns of system. sheep was once attributed to the testosterone in either intersex while a Results of cytogenetic analysis on absence of vascular anastomosis fluctuation characteristic of seasonal the caprine and ovine intersexes indi- between the fetuses sharing the uterus influence was evident in the ram and cate that both of these were blood (14). However, it is interesting to note the buck (10,17). chimeras. The exclusively XX make- that the occurrence of freemartin con- The absence of fluctuation in testo- up of the muscle tissue suggests that dition in goats was documented by sterone concentrations in the inter-

405 w.

Fig. 3. Histological sections of the gonad and gonaduct of the intersex goat. H&E. a: The left gonad showing seminiferous tubules (ST) of varied diameter and follicle like structures (arrows) without germ cells. X100. b: Another area of the left gonad showing degenerating follicle-like structures (arrows) and seminiferous tubules (ST). Note the abundance of small connective tissue cells (CC) surrounding a follicle-like structure. X50. c: The right gonad showing narrow seminiferous tubules and the abundant interstitium. X1W0. d: Epididymis of the right gonad from an area close to the vas deferens in normal males. X50.

406 Fig. 4. Histological sections of the uterine horn and the structures resembling the body of the uterus and seminal vesicle. a: The left uterine horn showing intact glands in low magnification (A) and in higher magnification (1). Note the inactive glands (G) and the flat epithelium (E). : X5O; a : X100. b: A section through the "body of the uterus" showing an abundance of submucosal glands (G). X5O. c: A higher magnification showing the tall epithelium (E) of the submucosal glands. Note the leukocyte infliltration (L) from the submucosal space towards the lumen. XIOO0. d: A section through the outer portion of the "uterine body" resembling the seminal vesicle. Note the concretions (Cn) in the lumen of the gland. XI100.

407 explanation for the high concentration of testosterone in the present cases could well be a decreased utilization of testosterone by the cells of the seminiferous epithelium. The absence offunc- tional germ cells and the inactive state of the seminiferous tubules may result in a reduced utilization oftestosterone which is considered to play a major role in the initial and terminal stages of spermatogenesis in normal males (18). Irrespective of the exact mechanism that leads to high testosterone concentration in circulation, the masculiniza- tion of the genitalia and the duct system in the male direction in both intersexes is unequivocal. Masculini- zation ofthe steroid dependent organs indicates that the target organs of these animals are sensitive to testosterone. The anatomical and behavioural characteristics ofthese freemartin type intersexes would make them excellent 3 teasers in a breeding program. ACKNOWLEDGMENTS The authors are grateful to Mr. Ed. R. Reyes, Mrs. Helen Randall, Mrs. Mary John, Mrs. Patricia Franks and Ms. Christine Stec for technical assis- tance. This work was supported in part by Natural Sciences and Engineering 5 Research Council of Canada and the Ontario Ministry of Agriculture and Food.

REFERENCES

1. EATON ON, SIMMONS VL. Herma- 6 phroditism in milk goats. J Hered 1939; 30: 261-266. 2. BIGGERS JD, McFEELY RA. Inter- sexuality in domestic animals. In: McLaren A, ed. Advances in reproductive physiology. London: Logos Press, 1966: 29-59. 3. HAMMERTON JL, DICKSON JM, POLLARD CE, GRIEVES SA, SHORT RV. Genetics of intersexuality in goats. J Reprod Fert (Suppi 7) 1969; 25-51. 4. DENNIS SM. Urogenital defects in sheep. Fig. 5. Diagram of the gross appearance of the genital tract of the intersex sheep. 1: gonads, 2: vas Vet Rec 1979; 105: 344-347. deferens, 3: ampulla, 4: seminal vesicles, 5: pelvic urethra, 6: clitoris. 5. BASRUR PK, COUBROUGH RI. Ana- tomical and cytological sex of a Saanen goat. Cytogenetics 1964; 3: 414-426. sexes described here, would appear to tion in plasma FSH and LH concen- 6. EATON ON. The relation between polled indicate that the gonadal testosterone trations in a freemartin ewe. These and hermaphroditic characteristics in dairy secretion of the intersexes is not under investigators hypothesized that the goats. Genetics 1945; 30: 59-61. the influence of the central nervous level of have 7. SHORT RV, HAMMERTON JL, high androgens may GRIEVES SA, POLLARD C. An intersex system. In this regard, it is worthy of "depressed LH to basal levels" without goat with a bilaterally asymmetrical repro- note that Saba et al (10) detected a abolishing"the spontaneous surges" in ductive tract. J. Reprod Fert 1968; 16: high concentration and erratic fluctua- gonadotrophins (10). An alternative 283-291Q.

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j,..... @ .. I .. 12. BOSU WTK, BARKER CAV. Steroid levels i ., I .., .4. F, after intramuscular injection of testosterone ',4 ii propionate in the caprine. Can J Comp Med 1982; 46: 390-394. 13. OHNO S, TRUJILLO JM, STENIUS C, ' . CHRISTIAN LC, TEPLITZ RL. Possible ,. *~~~~~~~~~~-.. germ cell chimeras among newborn dizy- .N:: }~~~~~ gotic twin calves (Bos taurus). Cytogenetics 1962; 1: 258-265. 14. LILLIE FR. The freemartin; a study of the action of sex hormones in the fetal life of cattle. J Exp Zool 1917; 23: 371-422. 15. ILBERY PLT, WILLIAMS D. Evidence of the freemartin condition in the goat. Wi Cytogenetics 1967; 6: 276-285. 16. SMITH MC, DUNN HO. Freemartin condition in a goat. J Am Vet Med Assoc 1981; 178: 736-737. 17. MUDNELI DS, SANFORD LM, PALMER WM, HOWLAND BE. Secre- tory patterns and circulation and seasonal

$ p changes in luteinizing hormone, follicle stimulating hormone, prolactin and testosterone in the male pygmy goat. J Anim Sci *wt 1979; 49: 543-553. 18. SETCHELL BP. Spermatogenesis and spermatozoa. Reproduction in mammals. -* - t Book 1. Second edition. Austin CR, Short RV, eds. London: Cambridge University A9 Press, 1982: 63-101. f.st

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9$ ..A .1: ..VS i:1 X,or 4-`~~~~~~~~~~~ .1 j=4-1. C: d Fig. 6. Histological sections of the gonads and gonaduct from the intersex sheep. a: Seminiferous tubules (ST) surrounded by abundant interstitial cells resembling fibroblasts and leukocytes (left gonad). X150. b: A higher magnification of the right gonad showing immature Leydig (L) cells and macrophages (M). X400. c: Cross sections of the epididymis showing tall epithelial cells enclosing a small lumen. Note the eosinophilic material in the lumen (Em). X100. d: Cross sections of the epididymis (above) showing hyperactive epithelium (HE) lining the lumen and the occlusion of the lumen with connective tissue cells (CC). X50.

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