Joanne M. Orth

Gonadal dysfunction in the spontaneously diabetic BB rat

Diabetes which occurs spontaneously in the BB Wistar rat is associated with reduced fertility, predominantly in breeding males. In the first month of diabetes, there is a significant (p less than 0.05) reduction in serum testosterone associated with a transient decrease of serum LH and the accumulation of lipid in Leydig cells. Between one and three months of diabetes, there is an increase in both serum testosterone and LH and a further deposition of lipid droplets in Leydig cells. From three to six months of diabetes, there is a reduction of serum testosterone similar to age-matched controls, but high serum LH levels persist. Similar levels of LH and testosterone are noted after six months of diabetes, and all BB rats show marked changes in seminiferous tubules. These morphological changes in tubules consist of increased tubular wall thickness, severe germ-cell depletion, and Sertoli-cell vacuolization. Similar morphological changes of testes associated with generalized atrophy are noted in all control rats after 16 months of age. Decreased fertility in the BB rat appears to be associated with a primary disorder of Leydig cells, which precedes changes in seminiferous tubules consistent with accelerated aging. Preliminary data in impotent diabetic men suggest that the BB rat may be a valuable model for investigating human diabetic impotence and infertility.

Analysis of Ppp1cc-Null Mice Suggests a Role for PP1gamma2 in Sperm Morphogenesis

Abstract Serine/threonine protein phosphatase 1 (PP1) consists of four ubiquitously expressed major isoforms, two of which, PP1gamma1 and PP1gamma2, are derived by alternative splicing of a single gene, Ppp1cc. PP1gamma2 is the most abundant isoform in the testis, and is a key regulator of sperm motility. Targeted disruption of the Ppp1cc gene causes male infertility in mice due to impaired spermiogenesis. This study was undertaken to determine the expression patterns of specific PP1 isoforms in testes of wild-type mice and to establish how the defects produced in Ppp1cc-null developing sperm are related to the loss of PP1gamma isoform expression. We observed that PP1gamma2 was prominently expressed in the cytoplasm of secondary spermatocytes and round spermatids as well as in elongating spermatids and testicular and epididymal spermatozoa, whereas its expression was weak or absent in spermatogonia, pachytene spermatocytes, and interstitial cells. In contrast, a high level of PP1gamma1 expression was observed in interstitial cells, whereas much weaker expression was observed in all stages of spermatogenesis. Another PP1 isoform, PP1alpha, was predominant in spermatogonia, pachytene spermatocytes, and interstitial cells. Examining the temporal expression of PP1 enzymes in testes revealed a striking postnatal increase in PP1gamma2 levels compared with other isoforms. Testicular sperm tails from Ppp1cc-null mice showed malformed mitochondrial sheaths and extra outer dense fibers in both the middle and principal pieces. These data suggest that in addition to its previously documented role in motility, PP1gamma2 is involved in sperm tail morphogenesis.

Gonocytes in testes of neonatal rats express the c-kit gene.

Information gathered from mutant mouse models and from studies on normal puberal and adult animals points to the product of the c‐kit gene, a tyrosine kinase surface receptor, and the kit‐ligand (KL) as important for gametogenesis in males. In fetuses, KL serves as a survival factor for primordial germ cells, at least in vitro, and in adults activity of the c‐kit gene has been indirectly related to survival and subsequent development of differentiating spermatogonia. However, because of the structural complexity of the seminiferous epithelium in adults, c‐kit mRNA has not yet been definitively localized to one or more types of spermatogenic cells. In addition, no information is currently available regarding the possible involvement of the c‐kit protein and its ligand in mediating germ cell development and/or Sertoli‐germ cell interactions immediately after birth when events critical for later onset of spermatogenesis are ongoing. Thus, the aims of the current study were (1) to determine whether the c‐kit gene is expressed in testes of neonatal and adult rats and, if so, by what specific cell types, and (2) to determine if those cells expressing the gene also produce the c‐kit receptor protein. For this, we isolated total RNA from testes of pups aged days 1–5 and from adult rat testes, and probed for the presence of c‐kit mRNA with Northern analysis. We identified the cells containing the c‐kit message by carrying out in situ hybridization with digoxigeninlabeled probes, thus allowing the colorimetric signal to be assigned beyond doubt to individual cells in sections of testes. We also utilized Western analysis and immunolocalization to confirm the presence of the c‐kit receptor protein in testes at these ages and to identify those cells types producing it. Our findings indicate that (1) neonatal gonocytes express the c‐kit gene and produce the receptor protein on postnatal days 1 through 5, spanning the time when they resume dividing and migrating, and (2) spermatogonia and, to a lesser extent, spermatocytes and spermatids of adults express the gene but c‐kit protein is present in detectable amounts only in spermatogonia and possibly a few early primary spermatocytes.

Use of in vitro systems to study male germ cell development in neonatal rats

The aim of this review is to summarize ways in which in vitro approaches have allowed us to investigate several aspects of gametogenesis in the male. In our laboratory, we have established both organ culture and cell co-culture methodologies and applied them to questions focused on cellular and molecular events important for development of primitive spermatogonia, or gonocytes, in testes of neonatal rats. We have described their postnatal reinitiation of mitosis and their migration to the basal lamina in anticipation of basal compartment formation and, through use of these in vitro systems, we have identified several mechanisms regulating these processes. These include matrix influence on mitosis and migration, adhesive mechanisms active between gonocytes and Sertoli cells, and involvement of the Kit receptor on germ cells and its ligand from Sertoli cells in supporting gonocyte migration, as described below.

Sperm from mice carrying two t haplotypes do not possess a tyrosine phosphorylated form of hexokinase

Mouse sperm contain a tyrosine phosphorylated form of hexokinase type 1 (HK1; Kalab et al., 1994: J Biol Chem 269:3810–3817) that has properties consistent with an integral plasma membrane protein. Furthermore, this tyrosine phosphorylated form of HK1 has an extracellular domain and HK1 is localized to both the head and flagellum of nonpermeabilized cells (Visconti et al., 1995c). We have characterized HK1 in mature sperm from sterile tw32/tw5 mice (mutant sperm) that have defects in motility and sperm‐egg interaction (Johnson et al., 1995: Dev Biol 168:138–149). Immunoprecipitation of mouse sperm extracts with an antiserum made against purified rat brain HK1 demonstrates the presence of HK1 in mutant sperm. Various biochemical and immunofluorescence assays indicate that at least a portion of the HK1 present in these cells is an integral membrane protein with an extracellular domain located on the sperm head and flagellum. However, immunoblot analysis with anti‐phoshotyrosine antibodies demonstrates that HK1 in mutant sperm is not tyrosine phosphorylated. Northern blot and RT‐PCR analysis does not indicate any obvious abnormalities in the transcription of somatic or germ cell‐specific HK1 isoforms in mutant testes, and RFLP analysis of recombinant mice indicates that no genes specifying HK1 isoforms are located on chromosome 17. We have mapped the locus responsible for the lack of tyrosine phosphorylation of HK1 mutant sperm to the most proximal (to the centromere) of the four inversions within the t haplotype. A male sterility factor is located in this same inversion (Lyon, 1986: Cell 44:357–363). Since the mutant sperm are unable to complete fertilization, there could be a relationship between sterility and the lack of tyrosine phosphorylation of HK1 in these mutant sperm.

Autoradiographic Localization of FSH-Binding Sites on Sertoli Cells and Spermatogonia in Testes from Hypophysectomized Rats

It is now well established that follicle-stimulating hormone (FSH) acts primarily on the seminiferous tubule of the mammalian testis, and that receptors for this action are located on tubule plasma membranes. Specific FSH-binding sites have been demonstrated in isolated seminiferous tubules from immature and mature rats, and it has been shown that most of the binding activity resides in the plasma membrane fraction (Means and Vaitukaitis, 1972; Means, 1973). The biochemical events that are initiated by the binding of FSH to its receptor are also well characterized. Incubation of FSH with either seminiferous tubules (Dorrington et al., 1972) or plasma membranes isolated from tubules (Means, 1975) results in an elevation of cyclic AMP (cAMP) levels that is hormone-and tissue-specific. The cAMP seems to act as an intracellular messenger, causing activation of cAMP-dependent protein kinase and subsequent transcriptional and translational events (Means, 1975).

Development of Postnatal Gonocytes In Vivo and In Vitro

Sperm production in the mature testis is the result of complex processes that have been the focus of intense study in recent years, resulting in an increased understanding of the mechanisms underlying spermatogenesis. However, far less information is available concerning the early postnatal development of the testis when a number of events occur that are critical for the later onset of spermatogenesis, including the resumption of germ cell mitosis and the arrival of some of these cells at the basement membrane.

Gonocyte—Sertoli Cell Interactions in Testes of Neonatal Rats

Our understanding of spermatogenesis in the adult testis has increased substantially in past years, as more and more investigators have focused on the role of interactions between somatic and germ cells in supporting this process. We now recognize, for example, the way in which adhesive interactions between Sertoli and germ cells control movement of young spermatocytes from the basal into the adluminal compartment and the critical support provided to the adluminal cohort of spermatogenic cells by the Sertoli cells. Considerably less is known, however, about how spermatogonia begin their passage through spermatogenesis and even less is understood about the ancestors of these cells, the gonocytes, and how they mature during the prespermatogenic period of testicular development.