Efficiency of Spermatogenesis: a Comparative Approach

Animal Reproduction Science 60±61Ž. 2000 471±480 www.elsevier.comrlocateranireprosci

Efficiency of spermatogenesis: a comparative approach

L. Johnson a,c,), D.D. Varner b, M.E. Roberts a, T.L. Smith a, G.E. Keillor a, W.L. Scrutchfield b a Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M UniÕersity, College Station, TX 77843-4458, USA b Department of Large Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M UniÕersity, College Station, TX 77843-4458, USA c Center for EnÕironmental and Rural Health, College of Veterinary Medicine, Texas A&M UniÕersity, College Station, TX 77843-4458, USA

Abstract

Efficiency of spermatogenesis is the estimated number of spermatozoa produced per day per gram of testicular parenchyma. Spermatogenesis is the process of cell division and cell differentiation by which spermatozoa are produced in testes. Efficiency of spermatogenesis is influenced by species differences in the numerical density of germ cell nuclei and in the life span of these cells. Activities of spermatogonia, spermatocytes, and spermatids partition spermatogenesis into three major divisionsŽ. spermatocytogenesis, meiosis, and spermiogenesis, respectively . Spermatocyto- genesis involves mitotic germ cell division to produce stem cells and primary spermatocytes. Meiosis involves duplication of chromosomes, exchange of genetic material, and two cell divisions that reduce the chromosome number and yield four spermatids. In spermiogenesis, spherical spermatids differentiate into mature spermatids which are released in the lumen of seminiferous tubules as spermatozoa. Spermatogenesis and germ cell degeneration can be quantified from numbers of germ cells in various developmental steps throughout spermatogenesis. Germ cell degeneration occurs throughout spermatogenesis; however, the greatest impact occurs during spermatocytogenesis and meiosis. There are species and seasonal influences on the developmental steps in spermatogenesis at which germ cell degeneration occurs. Number of Sertoli cells, amount of smooth endoplasmic reticulum of Leydig cells, and the number of missing generations of germ cells within the spermatogenic stage of the cycle influence efficiency of spermatogenesis. Efficiency of spermatogenesis is influenced to the amount of germ cell degenera-

) Corresponding author. Tel.: q1-979-845-9279; fax: q1-979-847-8981. E-mail address: [email protected]Ž. L. Johnson .

0378-4320r00r$ - see front matter q 2000 Published by Elsevier Science B.V. All rights reserved. PII: S0378-4320Ž. 00 00108-1 472 L. Johnson et al.rAnimal Reproduction Science 60±61() 2000 471±480 tion, pubertal development, season of the year, and aging of humans and animals. q 2000 Published by Elsevier Science B.V. All rights reserved.

Keywords: Spermatogenesis; Species comparisons; Germ cell degeneration

1. Introduction

1.1. Definition and background

Spermatogenesis is the sum total of the events that occur within the testis that produce spermatozoaŽ. Johnson, 1991b . Spermatogenesis occurs within seminiferous tubules of the testis. It is a lengthy, chronological process by which stem cell spermatogonia divide by mitosis to maintain their own numbers and to cyclically produce primary spermatocytes that undergo meiosis to produce haploid spermatids which differentiateŽ. without further division into spermatozoa. The efficiency of spermatogenesis is the number of spermatozoa produced per gram of testicular parenchyma and is not influenced by difference in testicular size among animals. Spermatocytogenesis, meiosis, and spermiogenesis are characterized by division andror differentiation of spermatogonia, spermatocytes, and spermatids, respectively, and are three major divisions of spermatogenesisŽ. Fig. 1 . In the bull, these divisions take 21, 23, and 17 days, respectively, for a total duration of 61 daysŽ Fig. 1; Amann, 1970. . During spermatocytogenesis, stem cell spermatogonia divide by mitosis to produce other stem cells that continue the lineage throughout the adult life of malesŽ Fig. 1. . Stem cells give rise to spermatogonia that cyclically produce committed spermatogonia which proliferate andror differentiate to produce primary spermatocytes that undergo meiosis. Meiosis allows exchange of genetic material between homologous chromosomes of primary spermatocytes and the production of haploid spermatidsŽ Fig. 1.Ž. . During spermiogenesis Fig. 1 , spermatids differentiate from cells with spherical nuclei into mature germ cells shaped like spermatozoa for that species. The flagellum is developed into a tail, and the head of the spermatozoon is composed of the compressed nucleusŽ. source of the male genome and an acrosome with its enzymes necessary to penetrate the layers of the egg.

1.2. Kinetics of spermatogenesis

The spermatogenic cycleŽ. cycle of the seminiferous epithelium is superimposed on the major divisions of spermatogenesisŽ spermatocytogenesis, meiosis, and spermiogenesis; Fig. 1. . The cycle of the seminiferous epithelium is ``a series of changes in a given areaŽ. region of seminiferous epithelium between two appearances of the same developmental stagesŽ.Ž steps '' Leblond and Clermont, 1952 . and lasts for 13.5 days in the bull. If spermiationŽ release of spermatozoa from seminiferous epithelium and counterpart to ovulation in the female. is used as a reference point, the spermatogenic cycle would be all the events that occur between two consecutive spermiations from a given region of the tubule. L. Johnson et al.rAnimal Reproduction Science 60±61() 2000 471±480 473

Fig. 1. Drawings and classification of germ cells at different developmental steps in the three major divisions of spermatogenesisŽ. spermatocytogenesis, meiosis, and spermiogenesis combined to make the eight stages of the cycle of bull seminiferous epithelium. During the 21 days of spermatocytogenesis, A spermatogoniaŽ. A enters cyclicŽ. at 13.5-day interval activity during stage III and undergo division to produce intermediate Ž. In , BŽ. B spermatogonia, and leptotene primary spermatocytes Ž. L . During the 23 days of meiosis, leptotene primary spermatocytes differentiate through zygoteneŽ. Z , pachytene Ž. P , and diplotene Ž. D before the first meiotic division to produce secondary spermatocytesŽ. SS , and the second meiotic division to produce Sa spermatidsŽ. Sa . During the 17 days of spermiogenesis, Sa spermatids differentiate through Sb11Ž. Sb , Sb 2 Ž.Sb21122 , Sc Ž. Sc , Sd Ž. Sd , and Sd Ž. Sd steps of development before spermiation as spermatozoa. The letters indicate the developmental step, and the numbers associated with each germ cell step indicate the developmental age of each cell type in the middle of each spermatogenic stage. The cycle length is 13.5 days, and the duration of spermatogenesis is 61 days in the bull. Modified from Johnson et al.Ž. 1994 .

The cycle length and frequency at which spermatozoa are released both are deter- mined by the rate at which committed spermatogonia enter the process of spermatogenesis. The cycle length and duration of spermatogenesisŽ from the production of committed spermatogonia to spermiation.Ž are species-specific Swierstra et al, 1974; Amann, 1986 . . The cycle length in days for the prairie vole is 7.2, hamster 8.7, mouse 8.9, rhesus monkey 9.5, rabbit 10.7, stallion 12.2, bull 13.5, beagle dog 13.6, and man 16 Ž.Clermont, 1963; Swierstra et al, 1974; Amann, 1981, 1986 . The stage represents an association of 4±5 germ cells, each of which is in a specific, chronological, developmental step in spermatogenesis. The assignment of stages repre- sent man-made divisions of naturally cyclically occurring cellular associations. In bulls, 474 L. Johnson et al.rAnimal Reproduction Science 60±61() 2000 471±480 the cycle has been divided into eight stagesŽ.Ž Amann, 1970 or 12 stages Berndtson and Desjardins, 1974.Ž. . Fourteen stages have been described in rats Clermont, 1972 , eight in the horseŽ. Swierstra et al, 1974 , and only six stages in humans Ž. Clermont, 1963 . The bullŽ.Ž Amann, 1970 and horse Swierstra et al, 1974 . , like most species, have mainly only one stage of the cycle represented in a cross-section of the seminiferous tubule. Humans have more than two stages per cross-sectionŽ. Clermont, 1963 .

1.3. Testis

Quantitative approaches have been extended to calculate daily sperm production, a quantitative measure of spermatogenesis to express the total number of spermatozoa produced per day by a testis or paired testesŽ Kennelly and Foote, 1964; Amann, 1970; Johnson, 1986b. . Considering the life span and theoretical yield of a specific germ cell, a daily expression of spermatozoan production can be obtained from the number of germ cells of that type in the testisŽ. Kennelly and Foote, 1964; Amann, 1970 . The life span of a germ cell is the duration of stages of the cycle in which that cell type occurs. Theoretical yields are calculated by 2 n, where n is the number of cell divisions between that cell type and spermatids. Daily sperm production per gram of testicular parenchyma is a measure of efficiency of spermatogenesis, and it is useful in species comparisonsŽ Fig. 2; Amann et al, 1976;

Fig. 2. Efficiency of spermatogenesis in various species based on potential daily sperm production per gram parenchyma at different developmental steps in spermatogenesis of the rat, bull, horse, boar, and human. Potential daily sperm production per gram is calculated from numbers of B spermatogonia, pachytene primary spermatocytes, Golgi and cap phase spermatidsŽ. round spermatids , and maturation-phase spermatids Ž elon- gated spermatids.Ž. . Adult rats )400 g experienced no significant loss during these different steps in spermatogenesis. Adult horses had early germ cell degeneration in spermatogenesisŽ end of spermatocyte formation. with no subsequent losses. Younger adult humans had significant losses during the second meiotic division. Humans and bulls have much lower efficiency of spermatogenesis than do other species. Boars and humans have significant losses in potential production at the end of meiosis. Bulls and horses have no significant losses at the end of meiosis. Modified from Johnson et al.Ž.Ž. 1981, 1984a,b, 1994 , Johnson 1986b , and Okwun et al.Ž. 1996 . L. Johnson et al.rAnimal Reproduction Science 60±61() 2000 471±480 475

Johnson, 1986b.Ž . Humans have a much lower efficiency 4±6=106rg. of spermatogen- esisŽ. Johnson et al., 1981 than do other species tested, including rhesus monkeys Ž23=106rg.Ž , rabbits 25=106rg.Ž , rats 20±24=106rg.Ž boars 23=106rg. , stal- lionsŽ 16±19=106rg.Ž , rams 21=106rg.Ž , hamsters 24=106rg. , and even bulls Ž12=106rg.Ž Amann et al, 1976; Amann, 1981; Johnson, 1986b . . Comparisons of daily sperm production per gram parenchyma vs. potential daily sperm production based on germ cells in different developmental steps of spermatogenesis facilitate detection of species differences in sites of germ cell degeneration in spermatogenesisŽ Fig. 2; Johnson et al, 1999a,b. . Lower efficiency of spermatogenesis in human testesŽ. compared to other species results from a longer duration of spermatogenesisŽ. 74 days , longer cycle length Ž 16 days.Ž Clermont, 1972 . , and lower density of germ cells in human testes Ž Johnson, 1986b. . In humans, the number of round spermatids per gram parenchyma is 49.2=106 Ž.Johnson, 1986a,b . In comparison, in the rat, whose cycle length is 12.9 days, and the horse whose cycle is 12.2 days, the number of round spermatids per gram is 190=106 Ž.Johnson et al, 1984a and 162=106 , respectivelyŽ. Johnson, 1985 . The percentages of the human testis occupied by seminiferous tubules and seminiferous epithelium are lower than that for bulls, horses, or ratsŽ. Fig. 3; Johnson, 1986b . The reason humans differ from other species in their lower testicular germ cell density and longer cycle length remains a mystery. However, it does not appear to be a problem for the survival of the human species, considering the population explosion on the earth. Germ cell degeneration at specific developmental steps of spermatogenesis has been quantified by comparing daily sperm production per gram parenchyma based on germ cell types in different steps of developmentŽ. Fig. 2; Johnson, 1986b . This has been done in the boarŽ.Ž.Ž. Kennelly and Foote, 1964 , bull, Amann, 1970 , rat Johnson et al, 1984a , horseŽ.Ž Johnson, 1985 , and human Johnson, 1986b . . However, among breeds of boars, the efficiency of spermatogenesis and potential daily sperm production based on germ

Fig. 3. Composition of testes in various species expressed as the percentageŽ. volume density of the testicular parenchyma occupied by seminiferous tubules or seminiferous epithelium. In the human, the volume density of seminiferous epithelium is less than 50% of the testicular parenchyma. Modified from JohnsonŽ. 1986b and Johnson et al.Ž. 1994 . 476 L. Johnson et al.rAnimal Reproduction Science 60±61() 2000 471±480 cell at different steps of spermatogenesis are similarŽ. Fig. 2 and reflect similar degenerative rates throughout spermatogenesis. For species comparisons, daily sperm production has been estimated at different development steps throughout spermatogenesisŽ. Fig. 2 . While humans and boars experienced a 30±40% reduction in potential sperm production during the end of meiosis, there was no comparable loss or degeneration during meiosis of the bull or stallion. In the breeding season of the stallion, there was a larger number of A spermatogonia than whose progeny could be sustained. This resulted in significant degeneration of B spermatogonia at the end of meiosisŽ. Johnson, 1985 . In the bull, a significant number of degenerated germ cells have been noted during spermatocytogene- sisŽ. Berndtson and Desjardins, 1974 . Another way to evaluate degeneration of germ cells at different developmental steps is by the ratio of more advanced germ cell per type A spermatogonium.

1.4. Season

StallionsŽ. unlike bulls, boars, rats, and humans modulate or regulate daily sperm production with season while continuing to produce spermatozoa throughout the year. Germ cell degeneration during meiosis and seasonal modulation of the number of A spermatogoniaw which is twice as large in the breeding season as in the non-breeding season of the horse testisŽ. Johnson, 1986bx are mechanisms that seasonally regulate spermatogenesis. The number of A plus B1 spermatogonia per testis in the breeding seasonŽ 5.1"0.2=109.Ž. was 71% higher p-0.01 than the number in the non-breeding seasonŽ 3.0"0.3=109.Ž. . Daily sperm production per testis was 84% p-0.01 higher in the breeding season. Season affects the number of different subtypes of spermatogonia per testisŽ. Fig. 11 . Seasonal modulation of A spermatogonia in a species may result from proliferation of renewing stem cells. The reserve stem cell spermatogonium is the youngest form of germ cells which may be dormant in testes active in spermatogenesisŽ Ao stem cell; Clermont, 1972. or actively involved in producing other stem cells or proliferating spermatogoniaŽ. As stem cells; Huckins, 1971 . These cells carry on the lineage throughout the life of adult males. Seasonal variation in the number of renewing stem cells has been found in other seasonal breeders such as rams and red deer stags Ž.Hochereau-de Reviers, 1981 . In the horse, the number of the most primitive spermato- - goniaŽ. A1 was 25% greater Žp 0.05 . in the breeding season Ž Fig. 4 . . Hence, seasonal differences in number of more primitive spermatogonia contribute significantly to seasonal differences in total number of spermatogonia in the horse. Season influences the developmental steps or spermatogonial subtypes that degener- r r ate. The yield of B21AorB 22A was greater in the breeding season of the horseŽ Fig. 4; Johnson, 1991a. . However, the yield of conversions of B2 spermatogonia to early primary spermatocytes was greater in the non-breeding season. The greater degeneration of B2 spermatogonia in the breeding season results form an overpopulation of A spermatogonia beyond the increased number of Sertoli cells in the breeding season. ResearchersŽ. Johnson, 1985, 1986a found a positive relationship between the number of

A1 spermatogonia and the amount of degeneration that occurred in A23 and A L. Johnson et al.rAnimal Reproduction Science 60±61() 2000 471±480 477

Fig. 4. Number and yield of spermatogonia in adult equine testes in the breeding and nonbreeding seasons. The number of horse A1231 , A , A , B , and B 2 spermatogoniaŽ. A 1231 , A , A B , and B 2 , respectively per testis and the yield of specific spermatogonial subtypes to B2 spermatogonia are different between the breeding or - non-breeding seasons.Ž. a The number of A1 spermatogonia is 25% Žp 0.05 . greater in the breeding season r r of the horse. The numbers of A231 , A , B , and B 2 spermatogonia and preleptotene leptotene zygotene primary spermatocytesŽ. PS are 39%, 83%, 91%, 110%, and 49% greater Žp-0.01 . , respectively, in the ) breeding season.Ž. b Although the conversions of A32 to B and A 12 to B spermatogonia are similar Ž.p 0.05 - between seasons, the conversions of A12 to B and A 22 to B are greater Ž.p 0.01 in the breeding season. The - conversion of B2 to early primary spermatocytes is less Ž.p 0.01 in the breeding season Ž from Johnson et al., 1991. . spermatogonia. As the result of a higher yield of early spermatogonial subtypes in the breeding season, the number of late spermatogonial subtypes was significantly increased Ž.Fig. 4 . This increased yield early in spermatogenesis appeared to make the greatest contribution to the significantly increased spermatogonial numbers in the breeding season. Although not seasonal, the bull had a significant loss of potential daily sperm productionŽ. regulatory degeneration or trimming of spermatogonial progeny between spermatocytogenesis and meiosisŽ. Fig. 2 .

Fig. 5. Effect of season on the number of Sertoli cells in the equine testis as viewed at different times of the year. Number of Sertoli cells found in 43±48 adult horses during each 3-month period throughout 1 complete year illustrates more Ž.p-0.05 Sertoli cells per gram parenchyma in May±July Ž the natural breeding season of the horse. than in other periods. The number of Sertoli cells per testis is greater in May±July compared with the value in August±October or February±April Ž.p-0.05 or compared with the value for November±January Ž.Žp-0.01 from Johnson and Nguyen, 1983 . . 478 L. Johnson et al.rAnimal Reproduction Science 60±61() 2000 471±480

Fig. 6. Species comparison in the number of Sertoli cells and in the germ cell:Sertoli cell ratio.Ž. a The number of Sertoli cells per gram parenchyma or per testis for the rat, bull, boar, horse, and human, andŽ. b the number of germ cells per Sertoli cell. The bull and human have fewer germ cells supported by each Sertoli cell than does the rat, horse, or boarŽ. from Johnson, 1986b; Johnson et al., 1994, 1996b; Okwun et al., 1996 .

Stereological methods of histologic images have illustrated seasonal variation in number of Sertoli cells and the germ cell:Sertoli cell ratio in the horseŽ Figs. 5 and 6; Johnson, 1986a.Ž . Seasonal variation in number of Sertoli cells Johnson and Thompson, 1983; Johnson, 1986a. have characterized the annual cycle of the Sertoli cell population in the horseŽ. Fig. 5 . There is significantly more Sertoli cells in the summer Ž natural breeding season.Ž. , and there is a dose month effect based on the time of the year the samples were takenŽ. Fig. 5 . The ratio of spermatogonia, spermatids, or all germ cells in stage VIII tubules was greater in the breeding season of the horseŽ. Johnson, 1986a . Hence, Sertoli cell function appears to be enhanced during the breeding season with seasonal modulation of spermatogenesis in the horse.

2. Germ cell degeneration

2.1. General considerations

Degeneration of germ cell occurs largely during spermatocytogenesis and meiosis Ž.Johnson, 1986b . Losses during spermatocytogenesis include 25% in mice, 11% in Sherman rats, and 75% in adult Sprague±Dawley rats. Greater degeneration of spermatogonia in rams occurs following long day illuminationŽ. Johnson, 1986b . Degenera- tion of B spermatogonia in horses is greater in the breeding season when the number of A spermatogonia is doubledŽ. Fig. 4; Johnson, 1985 . Meiotic divisions account for a 13% loss of potential production in mice, 2% in Sprague±Dawley rats, 27% in Sherman rats, 25% in rabbits, and 6±15% in stallions depending upon seasonŽ Johnson, 1985, 1986b.Ž. . In rams, fewer 40±50% spermatids were found after long day illumination Ž.Johnson, 1986b, 1991b . Germ cell degeneration during spermiogenesis was noted in Sherman rats and mice. A loss of 6% or lessŽ.Ž. depending on season occurred in horses Johnson, 1985 . Bulls Ž.Amann, 1970 and adult Sprague±Dawley rats Ž.)400 g had no significant germ cell loss during spermiogenesisŽ. Fig. 2; Johnson, 1986b . L. Johnson et al.rAnimal Reproduction Science 60±61() 2000 471±480 479

No significant degeneration of human germ cells occurred between type B spermatogonia and secondary spermatocytes or during spermiogenesisŽ. Fig. 2; Johnson, 1986b . A 30±40% loss from germ cell degeneration occurred during the meiotic divisions in humans. The loss of spermatozoan production late in meiosis is significantly, negatively correlated Ž.rsy0.86 with daily sperm production per gram parenchyma in humans. Likewise, serum concentrations of FSH in men are positively correlated with the percentage of germ cell degeneration during post prophase of meiosis. Degeneration detected late in meiosis occurred during the second meiotic division in young menŽ Fig. 2. and is greater in aged men. It is not known why humans have a greater degeneration rate late in meiosis than other speciesŽ. Fig. 2 , but it appears to be a critical step in human spermatogenesis that could be exploited to increase efficiency of spermatogenesis or to devise contraceptive strategies. There was no similar loss during meiosis in bulls, horses, or rats, but boars have a significant loss of potential for sperm production during post prophase of meiosisŽ. Fig. 2 .

3. Conclusions

Spermatogenesis is a long but orderly process by which spermatozoa are produced in seminiferous tubules and is divided into spermatocytogenesis, meiosis, and spermiogenesis. Germ cell degeneration occurs throughout spermatocytogenesis, but is greater during spermatocytogenesis and meiosis and can vary with pubertal development, age, and species. Bulls have a lower efficiency of spermatogenesis than most species including rats and horses examined, but higher than that of humans. Sertoli cell number is important in determining daily sperm production in bulls and boars as well as in other species including rats, horses, and humans.

Acknowledgements

The authors would like to thank Vince B. Hardy and Rebecca S. Heck for their excellent technical assistance and Penny Churchill for expert secretarial assistance with the manuscript. This work is funded in part by Link Estate Equine Endowment and NIH Funding K04AG00465, N01HD-83281, P30E09196, and T32G507273.

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