Maturation of Bovine Female Genitalia from Birth through Puberty C. Desjardins and H. D. Hafs J ANIM SCI 1969, 28:502-507.

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M A T U R A T I O N OF B O V I N E F E M A L E G E N I T A L I A F R O M BIRTH THROUGH PUBERTY 1

C. D E S J A R D I N S 2 AND H . D. HAFS a

Michigan State University, East Lansing

T R I K I N G changes in morphology of feS male genitalia, which occur during puberty, have been described in cattle (Sorensen fields in each of three sections from each of these tissues from every heifer. The remaining uterine, cervical and vaginal tissues were stored in 0.25M sucrose at - - 2 0 ~ C. for analyses of D N A and RNA (Tucker, 1964) and protein (Gornall et al., 1949). R e s u l t s and D i s c u s s i o n

et al., 1959). The results of such studies demonstrated the marked extent to which sexual maturation is influenced by level of feeding. However, to our knowledge, changes in reproductive organ DNA, RNA and protein with sexual maturation have been investigated only in rats (Desjardins et al., 1968). Those investigators quantified the rapid biochemical changes in reproductive organs of rats during puberty and delimited puberty on the basis of cellular function. The principal objective of the present research was to determine nucleic acids and protein as indices of growth and function of the tubular genitalia of heifers from birth through puberty. A second objective of this study was to determine functional relationships between reproductive organ growth during puberty and concurrent changes in pituitary levels of L H and FSH in the same heifers. D a t a on nutritional management, body growth, occurrence of estrus and pituitary gonadotropins in these heifers have been published (Desjardins and Hafs, 1968).

Ovarian weight increased nearly four times more rapidly than bodyweight from birth to 5 too. (table 1), but plateaued from 5 to 8 too. and resumed growth comparable to that for the body from 8 to 12 too. of age. This result is more apparent in figure 1, where increments in weight are expressed as multiples of weight at birth as Desjardins et al. (1968) did. With this method of relative growth analysis, weights of the various organs may be compared directly with each other and with bodyweight because differences in units are eliminated. That ovarian weight increases proportionately more rapidly than bodyweight of the heifer is in contrast to ovarian growth in rats which parallels bodyweight well past puberty (Desjardins et al., 1968). No follicles were visible macroscopically on the ovaries of heifers at birth (1 to 3 days Materials and M e t h o d s of age), but numbers of small and large Five Holstein heifers, from registered sires ovarian follicles increased to maxima at 4 mo., and production tested dams, were killed at decreased from 4 to 8 too., and remained relatively constant thereafter (table 1). These each month of age from birth to 12 months. observations suggest that the rapid increase The length and weight of the oviducts, uterus, cervix and vagina (terminated posteriorly at in ovarian weight from birth to 5 too. was the sub-urethral diverticulum) were deter- probably due in large part to increased nummined within 15 to 25 minutes. Number of bers of vesicular follicles. The plateau in follicles and corpora lutea on the surface of ovarian weight from 5 to 8 too. probably the ovaries were recorded. Tissues from the reflects continued growth of the ovarian interstitium with concurrent reduction in numupper one-third of the oviduct, the junction of the body and a horn of the uterus, the bers of vesicular follicles. But the data at 4 cervix and the anterior one-third of the vagina too. of age do not fit this relationship because were fixed in Bouin's solution for histological the number of follicles was greatest at this measurements. Epithelium heights were meas- age when ovarian weight was slightly reduced. ured in duplicate in each of five microscopic The large number of follicles at 4 too. suggests that secretion from them may be associated with subsequent onset of estrous cycles. 1 Published with the approval of the Director of the Experiment Station as Paper No. 4524. This research was supported Whether the follicles at 4 too. of age secrete by U.S.P.H.S. grant HD-01374. Present address: Department o~ Physiology and Pharmacolsteroid hormones is n o t known although ogy, Oklahoma State University, Stillwater. Roberts and Warren (1964) suggested that Animal Reproduction Laboratory, Dairy Department. 502

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BOVINE FEMALE GENITALIA

TABLE 1. B O D Y W E I G H T AND SOME OVARIAN CHARACTERISTICS FROM BIRTH TO 12 MO. OF AGE Body weight kg. 36.7 48.3 69.9 91.6 101.9 143.3 178.5 186.8 207.3 228.5 282.6 286.7 329.7 Paired ovarian wt. gm. 0.5-----0.1 ~ 1.2-----0.1 3.2-~-0.4 4.2___0.6 3.5+0.6 7.1 6.8-----1.8 6.9 7.i___+1.0 9.0-----1.5 9.4+--0.8 12.6-----3.0 12.4 No. of follicles ~ 5 ram. ~ 5 ram. 0 1.0
8.8

503

Age Mo. Birth 1 2 3 4 5 6 7 8 9 10 11 12

11.4 22.4 13.0 8.8 14.8 5.0 5.0 6.0 5.2 4.6

0 1.0 0 1.0 5.4 2.0 2.4 1.4 1.2 2.0 0.4 1.0 0.4

a Mean+standard error.

even the fetal bovine ovary is capable of some steroidal transformations. The second period of increase in ovarian weight (figure 1) occurred after estrous cycles were initiated (Desjardins and Hafs, 1968),

beginning during the seventh month of age. The reduction in number of follicles and the plateaued ovarian weight during the 4 too. before first estrus suggest that gonadotropin stimulation of the ovaries may have changed during this period. But pituitary content of follicle-stimulating hormone did not change significantly during this period. Although pituitary content of luteinizing hormone fluctuated greatly between 4 and 8 mo. (Desjardins and Hafs, 1968), no association between these pituitary criteria and the ovarian criteria reported here could be established. These observations do not exclude such a relationship, however, because Macmillan and Hafs (1968) found that changes in blood plasma levels of gonadotropins do not always parallel changes in pituitary levels of gonadotropin. Because this experiment was designed to obtain data at given ages, stage of estrous cycle at slaughter (table 2) was not considered a part of the design. All heifers previously observed in estrus possessed corpora lutea at slaughter and heifers not previously

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F i g u r e 1. C h a n g e s in b o d y a n d r e p r o d u c t i v e o r g a n w e i g h t s relative to t h o s e at birth.

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504

D E S J A R D I N S AND HAFS birth to 12 mo. were also proportionately greater than those for bodyweight. But the relative increase in D N A to 10 too. was only about two-thirds as great as the increases in uterine weight or RNA, suggesting hypertrophy of uterine cells concurrent with hyperplasia. Protein content of these uteri also supported this view because it was 26.0 times larger at 10 mo. than at birth. Relative to the values at birth (table 3) uterine weight (figure 1), RNA and protein increased more rapidly after 6 mo. than before this age, but the change in uterine DNA (presumably proportional to cell numbers) was less marked. These data suggest that uteri of heifers are influenced by significant levels of gonadal hormones during the seventh month of age before the onset of first estrus. These conclusions are in good agreement with similar conclusions for rats (Desjardins et al., 1968). The heifer uterine RNA and protein reached a plateau after 10 too. of age, suggesting that this age marks the end of rapid puberal growth of the uterus. Until 8 mo. of age, increases in weight, DNA, RNA and protein of the cervix were not as large as proportionate increases in bodyweight (table 4). In fact, increases in cervical DNA and RNA were less than those for bodyweight after 8 mo., during which period increases in cervical weight and protein became somewhat greater than the proportionate increases in bodyweight. Nevertheless, as was the case for uterine criteria, all cervical criteria increased more rapidly after 6 mo. of age than before (cervical weight, figure 1). Cervical D N A and RNA reached a plateau by 11 mo. of age. Vaginal weight increases (table 5) paralleled bodyweight increases to 6 too. and became slightly more rapid thereafter. Except

TABLE 2. DAY ~ OF ESTROUS CYCLE OF E A C H HEIFER SLAUGHTERED AFTER F I R S T ESTRUS Days from last observed estrus to slaughter 3, 4, 21 5, 20 7, 9, 12, 16 1, 1,3, 7, 12 2, 11, 13, 20, 23 5, 11, 17, 18, 19

Age Mo. 7 8 9 10 11 12 a Day of estrus was day 0.

found in estrus had none. Differences of this kind undoubtedly contributed to variation among heifers within an age for all reproductive criteria. For example, the small number of large follicles observed at 10 mo. (table 1) was probably caused by slaughtering four of five heifers within 7 days following estrus (table 2). Average weight of the oviducts increased proportionately to bodyweight from 0.5 gm. at birth to 2.8 gm. at 9 months. From 9 to 12 too., the increase in oviduct weight (3.3 gin. at 12 too.) was not as large as that for bodyweight. Oviduct length increased from 11.7 cm. at birth to 18.2 cm. at 5 mo. and to 21.7 cm. at 12 months. Changes in uterine weight and RNA with advancing age generally paralleled each other to 10 mo. of age (table 3). Except for a depression at 6 mo., both criteria increased proportionately more rapidly than bodyweight. For example, at 10 mo., bodyweight had increased 7.8-fold, whereas uterine weight had increased 22.5-fold (figure I). Uterine weight continued to increase after 10 mo., but uterine RNA did not. Increases in uterine D N A from

TABLE 3. SOME U T E R I N E C H A R A C T E R I S T I C S F R O M B I R T H TO 12 MO. OF AGE Age Mo. Birth 1 2 3 4 5 6 7 8 9 10 11 12 Wt. gm. 6 ~ 6-+__1 17-+3 24-+4-2 26-+3 41 4 25-+2 55-+10 58-+12 78-4-15 128-+8 132-4-7 150-+9 Length cm. 7.7--+0.3 6.1-+0.4 11.6-+0.6 11.7-+0.6 11.2-+0.5 15.8-+0.7 12.4-+1.1 15.1-+0.8 17.2-+0.6 16.7-4-1.2 20.5-+0.9 24.4-+0.7 24.3-+2.9 DNA mg. 38-+2 41-+2 87+11 119--+9 167-+22 186-+19 173-+12 282-+38 316__.52 376-+52 493-+123 435-+84 558_____44 RNA rag. 10+1 8-+1 28-+3 34-+2 52-+7 62 -+8 33-+2 99-+18 91-+24 121---+23 219-+53 180-+ 14 183-+ t0 Protein gin. 0.6-+0.1 0.7+0.1 1.6-+4-0.2 2.2 -+0.2 2.9-+0.3 3.7"4-0.4 2.6-+0.2 5.4-+0.9 6.0-+1.2 7.6 15.6-+-0.7 13.1-+0.9 I6.3-+0.9

a Mean+standard error.

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BOVINE FEMALE GENITALIA

TABLE 4. SOME CERVICAL C H A R A C T E R I S T I C S F R O M B I R T H TO 12 MO. OF AGE Age Mo. Birth 1 2 3 4 5 6 7 8 9 10 11 12 Wt. gm. 3.5 ~ 3.9+0.2 4.5+0.8 5.4~0.5 6.5+0.4 12.1+2.3 11.3 15.7+1.1 18.5 22.2+1.8 31.4+6.7 39.0+2.5 43.6 Length cm. 2.4 2.8+0.1 2.8---+0.2 3.4+0.2 2.1 3.3+0.2 3.4+0.2 4.0+0.3 4.2+0.5 4.4 3.9 4.9+0.3 4.9 DNA mg. 14+1 16+2 14__+2 16 23 ~---2 36 36~1 39m_4 47~8 50+7 66~13 95~13 82+9 RNA mg. 7___1 5~1 6+-1 8 10+1 21 17 20~2 21+2 32+6 55 65-+'4 61+6

505

Protein gm. 0.4+0.1 0.4~__0.1 0.5+0.1 0.6 0.8-+-0.1 1.3+0.3 1.4 1.8 2.2+0.3 2.4+0.2 4.1-+-0.9 4.5-+'0.2 5.5+0.7

a Mean+standard error.

for the extraordinary values at 10 too. of age, vaginal DNA and RNA both increased proportionately less than bodyweight. The significance of the very large values for vaginal criteria at 10 too. of age escapes us. It is tempting to associate them with the fact that four of these five heifers were between 1 and 7 days post-estrus at slaughter (table 2), but Hackett and Hafs (1969) found much smaller changes in the same vaginal criteria during the estrous cycle. Height of the luminal epithelium of reproductive organs revealed changes not found in the biochemical or in the other anatomical criteria. Epithelial cell height of uterus and cervix, and cell layer height of the vaginal epithelium were stimulated at birth and regressed after birth (table 6). After this regression, height of the epithelium of vagina, cervix and uterus did not recover to birth levels until 4 mo., 10 too. and 9 mo., respectively. Stimulated heights of the epithelium in these three organs at birth is probably caused by the very high maternal estrogen titers just before parturition (Mellin et al,

1966). A different pattern is evident for oviducal epithelial cell height which was reduced at 1 mo., but rebounded to birth levels by 2 mo. of age and increased no further. Thus, if height of oviducal epithelial cells is controlled by ovarian hormones, it is sensitive to much lower levels of these steroids than height of the epithelium in the other tubular genitalia. Another micromorphological difference between the epithelium of the oviduct and epithelia of the other tubular genitalia was the presence at the luminal surface of oviducal epithelia of dense staining structures which resembled cell nuclei in size, shape and tinctorial properties (figure 2). Sorensen et al. (1959) also observed these and suggested that they may be nuclei derived from epithelial cells and extruded into the oviducal lumen, but Asdell (1964) suggested that they may be globules of cytoplasm containing intensely basophilic centers. In any event, these unusual structures were most abundant in heifers slaughtered after their first estrus. They were virtually absent in heifers before

TABLE 5. SOME V A G I N A L C H A R A C T E R I S T I C S F R O M B I R T H TO 12 MO. OF AGE Age 3/[o. Birth 1 2 3 4 5 6 7 8 9 10 11 12 a Mean Wt. gin. 12 ~ 11+1 18+2 26 20+1 50~8 56 78 84+10 102 190 73 112~__14 115+8 error. Length cm. 5.6+0.3 9.9-+-0.5 7.9~0.5 9.2+0.8 6.7!-0.6 9.7+0.5 10.2 + 0 . 7 11.0~0.7 11.6~0.7 11.4 14.2 10.6+~0.6 10.2 ~+1.0 DNA mg. 38 31~__3 37+4 46 49+3 74 91 131+5 130+10 157---+12 300_89 158+19 150-+-15 RNA mg. 22 12+1 20+3 27 33 + 2 65+11 80~6 95 90+10 122~3 280__+95 151~24 146"+10 Protein gin. 1.4 1.4 2.2 2.9+0.5 2.6+0.1 5.5+0.8 6.6+0.5' 10.8+0.4 11.3~__1.3 12.4+0.9 23.7-+-9.1 14.0 14.7+___0.9

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506

DESJARDINS

AND

HAFS

F i g u r e 2. P h o t o m i c r o g r a p h s (X290) of o v i d u c t s ( t o p ) a n d uteri ( b o t t o m ) a t 5 too. (left) a n d 12 too. ( r i g h t ) . Oviducal epithelia possessed s e c o n d a r y folds at 5 too. a n d t e r t i a r y folds b y 12 m o n t h s . Note densely stained bodies w h i c h a p p e a r to be nuclei e x t r u d e d f r o m the epithelium a t 12 too. ( u p p e r r i g h t ) . E n d o m e t r i u m of u t e r u s a t 12 too. w a s sectioned t h r o u g h t h e wall of t h e n e c k a n d t h r o u g h t h e l u m e n of the basal p o r t i o n of a gland.

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BOVINE FEMALE GENITALIA TABLE 6. HEIGHT OF THE VAGINAL EPITHELIAL CELL LAYER AND OF THE CERVICAL, UTERINE AND OVIDUCAL EPITHIAL CELLS FROM B I R T H TO 12 MO. OF AGE
Epithelial height Age
~Om

507

Vagina 14.7+0.4a 6.1+0.3 5.4~0.3 8.9+0.6 15.3 18.2-t-0.8 12.5___+0.5 15.6+0.5 16.250.5 17.8~0.5 20,0~0.5 25.7 25.4_+1.1

Cervix 19.1~0.7 7.9+0.2

8.7+_0.2

Uterus 20'.9+-0.7 20.8~0.8 16,2~0.6 I7,1-;-0.3 11,5+0,.5 16.5~0.6 10.7___+0.2 20.1+0.8 14.8+__0.4 24.3~0.6 27.9~0,5 29.3~0.4 33.0~0.6

Oviduct 22.8+_0.7 15.5+0.3 30.5-4-0.3 30.6+0.3 31.4+0.3 30.2~-0.3 29,9 _+0.3 30.9-4-0.3 29.6+0.2 31.1~0.4 30.1~0,4 29.2+_0.2 28.4+0.2

Birth 1 2 3 4 5 6 7 8 9 10 11 12

9.4~0:.2 11.3 11.6+0.3 10.2~0.3 12,1+0.3 12.0+--0.3 lS.l 24.8+0.6 24.1~0,7 23.3~0.6

~ .~gean i~ m~crons+standard error.

6 mo. of age. T h e oviducal epithelium was always ciliated, even in heifers a t birth. However, subjective examinations suggested fewer cilia existed before than after first estrus. Similarly, microscopic inspection led to the conclusion t h a t secretion blebs at the luminal extremities of oviducal cells were much more common in animals after first estrus. Both superficial and basal uterine glands were absent until 5 too. of age, b u t t h e y became progressively larger and the epithelial cells lining them became more columnar after 6 mo. of age (figure 2). T h e cervical epithelium was pseudostratified at all ages, b u t numerous secretion blebs became especially conspicuous only during and after rapid puberal development between 6 and 10 too. of age. These micromorphological observations, in agreement with the biochemical and gross anatomical measurements, support the conclusion that rapid puberal growth begins during the seventh month of age in Holstein heifers. Several criteria plateaued and some were even reduced after 10 mo., suggesting t h a t this age m a y m a r k the end of rapid puberal development.

remained relatively constant thereafter. Gross anatomical and biochemical criteria of uteri, cervices and vaginae increased a t rates similar to r a t e of b o d y growth until 6 too. of age, but shifted to more rapid growth thereafter. These changes in rates of reproductive growth were most pronounced for organ weights, R N A content and protein content and less marked for D N A content. H e i g h t of the luminal epithelium of the tubular genitalia was stimulated at birth a n d regressed b y 1 or 2 mo. of age. Thereafter, increases in height of epithelia in vaginae, cervices and uteri were most rapid after 6 mo., similar to changes in organ weights. Further, secretion blebs on oviducal and cervical epithelia and bodies similar to nuclei a p p a r e n t l y extruded from oviducal epithelial ceils became more a b u n d a n t after 6 too. of age. These d a t a indicate t h a t r a p i d puberal growth in Holstein heifers commences during the seventh month of age and is largely terminated b y 10 too. because several of the measured criteria increased at reduced rates after this age. Literature Cited

Summary
Sixty-five Holstein heifers were killed at monthly age intervals in groups of five from birth through 12 too. of age to determine morphological and biochemical criteria of their reproductive organs. Ovarian weight increased 2.7 times faster than bodyweight. While no ovarian follicles were visible macroscopically a t birth, their numbers increased to a maxim u m at 4 mo., decreased to 8 mo. of age and

Asdell, S. A. 1964. Patterns of Mammalian Reproduction. (2nd ed.) Comstock Publishing Associates, Ithaca, N.Y. Desjardins, C. and H. D. Hafs. 1968. Levels of pituitary FSH and LH in heifers from birth through puberty. J. Animal Sci. 27:472. Desjardins, C., K. L. Macmillan and H. D. Hafs. 1958. Reproductive organ DNA and RNA of male and female rats from birth to 100 days of age. Anat. Rec. 161:17. Gornall, A. G., C. J. Bardawill and M. M. David. 1949. Determinations of serum proteins by means of the biuret reaction. J. Biol. Chem. 177:75I. Haekett, A. J. and H. D. Hafs. 1969. Biochemical and morpholo~cal changes in bovine tubular genitalia during the estrous cycle. J. Animal Sci. 28: in press. Macmillan, K. L. and H. D. Hafs. 1968. Pituitary and hypothalamic endocrine changes associated with reproductive development of Holstein bulls. r. Animal Sci. 27:1614. Mellin, T. N., R. E. Erb and V. L. Estergreen, Jr. 1966. Urinary excretion of estrogen by the bovine before and after parturition. J. Animal Sci. 25~955. Roberts, J. D. and J. C. Warren. 1964. Steroid biosynthesis in the fetal ovary. Endocr. 74:846. Sorensen, A. J., W. Hansel, W. H. Hough, D. T. Armstrong, K. McEntee and R. W. Bratton. 1959. Causes and prevention of reproductive failures in dairy cattle. I. Influence of underfeeding and overfeeding on growth and development of Holstein heifers. Cornell Univ. Agr. Exp. Sta. Bul. 935. Tucker, H. A. 1964. Influence of number of suckling young on nucleic acid content of lactating rat mammary glands. Proc. Soc. Exp. Biol. Med. 116: 218.

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