The goal of this morphometric study was to obtain quantitative information on the seminiferous tubules of Sprague-Dawley rats, including changes seen at various stages of the cycle of the seminiferous epithelium. Tissue from perfusion-fixed testes was embedded in Epon-Araldite; and sections were subjected to morphometric measurements at the light microscopic level, using point counting for volume densities and the Floderus equation for numerical densities. Changes occur in the diameter of the seminiferous tubule, as well as in the volume of the seminiferous epithelium and tubule lumen, from stage to stage during the cycle. A significant constriction of the seminiferous tubule accompanies spermiation. The volume of the seminiferous epithelium per unit length of the tubule begins to increase after stage XIV, and peaks at stage V of the next cycle. The tubule lumen increases dramatically from stages V to VII, at the expense of the epithelium. The number of Sertoli cells is constant per unit length of the seminiferous tubule at all stages of the cycle. This is also true for primary spermatocytes of various developmental phases and for round spermatids from step 1 through step 10 of spermiogenesis. The average number of younger (preleptotene, leptotene, zytgotene) primary spermatocytes per Sertoli cell is 2.34 +/- 0.082 (SEM), the number of older (pachytene, diplotene) primary spermatocytes per Sertoli cell is 2.37 +/- 0.064, and the ratio of step 1-10 spermatids to Sertoli cells is 7.89 +/- 0.27. By studying tangential views of serially sectioned seminiferous tubules at stage V, it is shown that the number of step-17 spermatids associated with each Sertoli cell averages 8.35 +/- 0.128, although the counts ranged from 6 to 11. The only appreciable occurrence of cell death after the last spermatogonial mitosis appears to be a 15% loss during the first meiotic division. From our morphometric results, corrected for volume changes during preparation for microscopy, there are 15.7 million (+/- 0.99 million) Sertoli cells per gram of fresh rat testis. The length of seminiferous tubule per gram of testis is estimated to be 12.4 +/- 0.56 meters, and the tubule surface area per gram testis is 119.7 +/- 2.57 cm2. The daily production of mature spermatids is 9.61 million (+/- 0.615 million) per gram of testis.

Hypophysectomy or sc implantation of testosterone-estradiol 17 beta (T-E) filled polydimethylsiloxane capsules for 5 days caused a dramatic reduction in testosterone secretion when testes subsequently were perfused in vitro. The diminution in testosterone-secreting capacity of testes from T-E treated rats was coupled closely with reductions in the membrane surface areas of Leydig cell cytoplasmic organelles, particularly those of the smooth endoplasmic reticulum. Simultaneous treatment of T-E implanted rats with LH (12 micrograms/day), but not with FSH, PRL, TSH, or GH, maintained both the Leydig-cell cytoplasmic membranes and the capacity of testes to secrete testosterone in vitro. Testosterone secretion by testes from hypophysectomized rats treated simultaneously with T-E plus LH was identical to that in control rats. Therefore, T-E did not inhibit directly the Leydig cell steroidogenic apparatus. Taken together these results suggest that one of the trophic effects of LH in the Leydig cell is to maintained the integrity of smooth endoplasmic reticulum and enzymes responsible for the conversion of pregnenolone to testosterone.

Depletion of endogenous LH with sc implants of testosterone-17 beta-estradiol (T-E) caused a reduction in the Leydig cell smooth endoplasmic reticulum (SER) over a 10-day treatment period. Decreases also occurred in some, but not all, of the testicular steroidogenic reactions responsible for the conversion of pregnenolone (PREG) to testosterone. The conversions of progesterone (PROG) to 17 alpha-hydroxyprogesterone, 17 alpha-hydroxyprogesterone to androstenedione, and androstenedione to testosterone were significantly correlated (P less than 0.05) with the loss of Leydig cell SER. In contrast, the testicular conversion of PREG to PROG in rats deprived of endogenous LH for up to 10 days was identical to that in intact controls. Similar results were obtained when rats were hypophysectomized for 10 days. These results indicate that the Leydig cell enzyme activities responsible for converting PREG to PROG are distributed in the Leydig cell SER fraction which remains in Leydig cell cytoplasm 10 days after LH withdrawal, and thus, the bulk of these enzyme activities are sequested in a SER compartment that is resistant to LH withdrawal.

The fine structure of the testicular interstitium was studied in normal adult golden hamsters sacrificed in the reproductive season (spring and summer) and in the winter. The Leydig cells in the reproductively active testes contain abundant endoplasmic reticulum (ER) and numerous mitochondria. The ER occurs in the form of flattened cisternae and tubules, the former prevailing. The cisternae are extremely extensive and are partly granular and partly agranular, their ends being continuous with the tubluar reticulum. Mitochondria intervening between the cisternae are closely associated with the agranular portions of the latter. Adjacent to the Golgi complex and continuous with the centrosome a unique filamentous body with a dense laminar core is often observed. In the regressive testes, the Leydig cells show a great reduction of cytoplasmic volume and a remarkable decline of the organelles, especially agranular tubules. The possible functional significance of the tubular and cisternal ER with the associated mitochondria is discussed in relation to the biosynthesis of androgens. Macrophages appear to constitute another important population of the interstitial cell clusters.

In the present study, we explored the restoration effects of exogenous LH on Leydig cell ultrastructure and testicular steroidogenesis in rats that were deprived of endogenous LH via treatment with testosterone-17 beta-estradiol-filled Silastic implants for 10 days. Exogenous LH was supplied continuously via Alzet miniosmotic pumps at the rate of 1 microgram/h for 3, 6, 12, and 24 h or 1, 2, 4, 8, and 12 days. Testes were then perfused in vitro with medium containing 1) LH, 2) 20 alpha-hydroxycholesterol, or 3) pregnenolone substrate, which allowed us to assess LH-stimulated testosterone secretion, cholesterol side-chain cleavage activity, or the conversion of pregnenolone to testosterone, respectively. Other testes were perfusion fixed via the testicular artery for morphometric measurement of Leydig cell number and volume per testis and the surface area of Leydig cell cytoplasmic smooth endoplasmic reticulum (SER), inner mitochondrial membrane, and outer mitochondrial membrane. The results verified that Leydig cell smooth endoplasmic reticulum and inner and outer mitochondrial membrane surface areas are drastically diminished (P less than 0.05 vs. intact controls) by LH withdrawal. Also, the results verified that exogenous LH administered in situ restores Leydig cell ultrastructure and capacity to biosynthesize testosterone. However, the recovery of Leydig cell structure and steroidogenic reactions occurred at strikingly different rates upon restoration of LH after 10 days of the treatment with testosterone-17 beta-estradiol implants. For example, the restoration of testicular capacity to synthesize progesterone in response to LH stimulation or 20 alpha-hydroxycholesterol substrate was completed within 24 h. In contrast, the restoration of Leydig cell SER and testicular capacity to synthesize testosterone from pregnenolone was completed only after 8 days of continuous LH treatment (P greater than 0.05 vs. intact controls). Thus, our results show that LH rapidly restores Leydig cell post-LH receptor steroidogenic events up to and including cholesterol side-chain cleavage activity. Interestingly, there is no temporal association between the recovery of cholesterol side-chain cleavage activity and the surface area of inner mitochondrial membrane surface area. In contrast, 8 days are required to coincidentally restore SER surface area and the capacity of Leydig cells to synthesize testosterone from pregnenolone. We conclude that different cellular mechanisms are involved in the LH-dependent restoration of inner mitochondrial cholesterol side-chain cleavage activity and SER-associated conversion of pregnenolone to testosterone.